Molecular Diagnostics: Promises and Possibilities

Molecular Diagnostics: Promises and Possibilities Mousumi Debnath · Godavarthi B.K.S. Prasad · Prakash S. Bisen Mole...

Author: Mousumi Debnath | Godavarthi B.K.S. Prasad | Prakash S. Bisen

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Molecular Diagnostics: Promises and Possibilities

Mousumi Debnath · Godavarthi B.K.S. Prasad · Prakash S. Bisen

Molecular Diagnostics: Promises and Possibilities

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Dr. Mousumi Debnath Jaipur Engineering College & Research Centre Dept. Biotechnology Jaipur-302022 India

Dr. Godavarthi B.K.S. Prasad Jiwaji University School of Studies in Biochemistry Gwalior-474011 India

Dr. Prakash S. Bisen Jiwaji University School of Studies in Biotechnology Gwalior-474011 India

ISBN 978-90-481-3260-7 e-ISBN 978-90-481-3261-4 DOI 10.1007/978-90-481-3261-4 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009941372 © Springer Science+Business Media B.V. 2010 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Molecular diagnostics represents a revolution postponed, perhaps, but one that is now clearly underway. Early and accurate diagnosis in medicine goes big way in prevention and treatment of communicable as well as non-communicable disorders. Traditional tests typically screen for a biochemical constituent or specific antibody or antigen derived from infectious agent to a particular disease. Molecular diagnostic tests typically analyze key DNA, RNA, or protein biomarkers (analytes) to identify a disease, determine its course, evaluate response to therapy, or predict individual predisposition to a disease. The techniques applied involve analysis of DNA sequences, DNA methylation patterns, gene expression profiles, proteins, protein expression, or combinations of these biomarkers. Such biomarkers provide direct information about genotypic and/or phenotypic changes associated with specific diseases or responses to treatment. Biomarker analysis has also become an important tool in drug discovery, preclinical drug development, and patient monitoring during clinical trials. The first few chapters of the book deals with the concepts of genomics and molecular biology. Specificity and sensitivity of DNA based tests are much higher, and the test is quicker than identification by traditional laboratory methods. In the case of human immunodeficiency virus (HIV), the initial detection limit in the late 1980s was 22 days post infection. However, the advent of nucleic acid testing for HIV RNA shortened this time to 11 days post infection. This highlights another distinct advantage offered by molecular diagnostics over immunoassays – amplification. The polymerase chain reaction (PCR) allows for the billion-fold amplification of available genetic material, thereby allowing the detection of minute amounts of genetic material and greatly increasing the sensitivity of the assay. No such amplification technique exists for proteins. A series of technologies developed over the years to address the challenges of molecular diagnostics include eight major areas: amplification technologies (gene and signal), blotting technologies, electrophoretic technologies, microarray technologies, probe technologies, restriction fragment-length polymorphism (RFLP) analysis, RNA inhibition analysis, and single nucleotide polymorphism (SNP) analysis and software. These developments have set the stage for excellent growth potential in molecular diagnostics.

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Preface

Current molecular diagnostics are primarily single-analyte tests involving the detection of a single gene or protein. However, many disease-related processes are multifactorial, involving the abnormal expression of multiple genes or proteins. Second-generation molecular diagnostics are anticipated to utilize novel detection technologies and multiplexing platforms to allow the measurement of a large number of analytes simultaneously. These innovations will increasingly utilize multiplexing platforms such as DNA microarrays that perform parallel biomarker analyses. Molecular diagnostics also has an additional ace up its sleeve – the capability to multiplex. A single assay can be used for the detection of multiple genetic targets. An example of this would be the simultaneous detection of HIV, hepatitis C and hepatitis B. This multiplexing capability helps save time, costs and represents a more effective diagnostic tool than an immunoassay for a single analyte. The next few chapters is focused on development of various nucleic acid technologies starting from PCR to microarrays. Molecular diagnostics got its big break following the successful sequencing of the human genome in 2003. DNA Testing has emerged fully from research into clinical practice and becoming a dominant platform in clinical medicine. This allowed the identification of portions of host or infectious agent genome that are responsible for disorder, as well as those that could be used as a marker to predict adverse reaction, dosing and efficacy to a particular drug. Applications in molecular diagnostics currently range from screening for infectious diseases and genetic disorders. It could test for genetic mutations and genome changes associated with cancers, or for the sequence or biomarker associated with a type of cancer. It has been particularly successful in diagnosing colorectal cancer, Methicillin-resistant Staphylococcus aureus (MRSA), breast cancer, respiratory viruses, prostate cancer and human papillomavirus (HPV). It could also be used to sequence individual genomes to identify a predisposition to developing disorders such as Alzheimer’s disease, Parkinson’s disease, diabetes and alcoholism. The molecular diagnostics is going to play an important role in pharmacogenomics, or personalized medicine. These methods allows to test an individual’s genetic disposition to a specific drug for most effective dosage and also helps to detect if the patient would show side effects. Molecular diagnosis aids in determining the right dosage of the right drug and at the right time for each patient. Molecular diagnostics is considered to work faster than traditional microbiology and more sensitive than traditional immunoassay testing. The capabilities of molecular diagnostics are increasingly being recognized due to global trends in emerging infectious diseases. Molecular assays have the capability to differentiate viral subtypes, detect genetic predispositions to a disease and monitor the course of a disease. The severe acute respiratory syndrome (SARS) corona virus and avian influenza outbreak in the recent past created an increasing awareness for this technology. The next few chapters of the book deals with the applications of molecular diagnostics in infectious, non-infectious and metabolic disorders. Semi and fully automated molecular testing platforms are being developed that address the issues of pre-analytical sample preparation and cross contamination. This will eventually allow molecular diagnostics to capture a larger segment of the

Preface

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infectious disease diagnostics market and achieve wider penetration in clinical diagnostics. The last chapter of the book deals with molecular diagnostics, its segments, market potential and current and future trends. The major shortcoming of molecular diagnostics lies in its high cost and the high level of technical skill needed to run complex assays. However, there is no match for the specificity and sensitivity of molecular assays. The adoption of the technology is increasing globally and the costs of molecular assays are expected to drop following the expiration of various PCR patents. Despite these obstacles, the speed and sensitivity of molecular diagnostics would gain more market share from traditional immunoassay testing, especially as costs come down. We thank Mr. Devendra Singh, Mr. Gulab Singh Thakur, Mr. Bhagwan S. Sanodiya, Mr. Vikas Dubey for their valuable assistance in preparation of this book. We are particularly indebted to Mr. Vikas Dubey, Web Designer, Vikrant Institute of Technology and Management, Gwalior for front cover image design. Finally we owe an unquantifiable debt to our families, who has seen the book built sentence by sentence and whose encouragement has never flagged. Jaipur, India Gwalior, India Gwalior, India

Mousumi Debnath Godavarthi B.K.S. Prasad Prakash S. Bisen

Contents

1 Introduction to Molecular Diagnostics . . . . . . . 1.1 Prologue . . . . . . . . . . . . . . . . . . . . 1.2 Clinical Diagnostics Entering a New Phase . . 1.3 Concept of Molecular Diagnostics . . . . . . 1.4 Molecular Diagnostic Technology and Health Care Industries . . . . . . . . . . . . . . . . 1.5 Molecular Diagnostic Approaches . . . . . . 1.5.1 Nucleic Acid Test Systems . . . . . 1.5.2 Gene-Based Diagnostics . . . . . . . 1.6 Conclusion . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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2 Omics Technology . . . . . . . . . . . . . 2.1 Prologue . . . . . . . . . . . . . . . 2.2 Concept of Omics . . . . . . . . . . 2.3 Genome and Genomics . . . . . . . 2.4 Transcriptome and Transcriptomics . 2.5 Proteome and Proteomics . . . . . . 2.6 Metabolome and Metabolomics . . . 2.7 Integration of Omics . . . . . . . . 2.8 Conclusion . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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3 Recombinant DNA Pharmaceuticals . . . . . . . . . . . 3.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . 3.2 Concept . . . . . . . . . . . . . . . . . . . . . . . 3.3 Tools of Recombinant DNA Technology . . . . . . 3.3.1 Common Enzymes Used in Recombinant DNA Technology . . . . . . . . . . . . . 3.3.2 Vectors . . . . . . . . . . . . . . . . . . . 3.3.3 Hosts . . . . . . . . . . . . . . . . . . . . 3.3.4 DNA Libraries . . . . . . . . . . . . . . . 3.3.5 Library Screening . . . . . . . . . . . . . 3.3.6 Polymerase Chain Reaction . . . . . . . .

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3.3.7 Analysis of Cloned DNA Sequences . . . . . 3.3.8 Expression of Cloned Genes . . . . . . . . . . 3.4 Therapeutic Applications of Recombinant DNA Derived Pharmaceuticals . . . . . . . . . . . . . . . . 3.4.1 Recombinant Therapeutic Proteins . . . . . . 3.4.2 Recombinant DNA Technology and Medicine 3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4 Aptamers: In Vitro DNA Selection . . . . . . . . . . . . . . . 4.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Selection of Aptamers . . . . . . . . . . . . . . . . . . . 4.4 Applications of DNA and RNA Aptamers . . . . . . . . 4.4.1 Aptamers for Detection of Pathogens . . . . . . 4.4.2 Aptamers for Therapy . . . . . . . . . . . . . . 4.4.3 Aptamers to Detect Change in Gene Expression 4.4.4 Aptamers as Diagnostics and Therapeutics . . . 4.4.5 Aptamers as Radiopharmaceutical Tools . . . . 4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5 Reporter Gene . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Reporter Gene Assay . . . . . . . . . . . . . . . . . . 5.3.1 Luc Reporter Gene System . . . . . . . . . . 5.3.2 Ruc Reporter Gene System . . . . . . . . . . 5.3.3 Green Fluorescent Protein (GFP) Reporter Gene System . . . . . . . . . . . . . . . . . . 5.3.4 Human Growth Hormone (hGH) Reporter Gene System . . . . . . . . . . . . . . . . . . 5.3.5 Beta-Galactosidase Reporter Gene System . . 5.3.6 CAT Reporter Gene System . . . . . . . . . . 5.3.7 LacZ Reporter Gene System . . . . . . . . . . 5.3.8 GUS Reporter Gene System . . . . . . . . . . 5.4 Transformation and Transfection Assays . . . . . . . . 5.5 Gene Expression Assays . . . . . . . . . . . . . . . . 5.6 Promoter Assays . . . . . . . . . . . . . . . . . . . . 5.7 Role of Reporter Gene Assay in Molecular Diagnostics 5.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6 Impact of HGP on Molecular Diagnostics . . . . . . . . . . . . . . 6.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Origin of the Human Genome Project . . . . . . . . . . . . .

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6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10

Goals and Issues of HGP . . . . . . . . . . . . . . . . . . . . The Human Genome and Genetic Variation . . . . . . . . . . Human Genome Project and Health Services . . . . . . . . . . Single Gene Disease . . . . . . . . . . . . . . . . . . . . . . New and Individualised Drug Treatments . . . . . . . . . . . The Achievements and Challenges of the HGP . . . . . . . . . Information Transfer . . . . . . . . . . . . . . . . . . . . . . Impact of the Human Genome Project on Epidemiologic Research . . . . . . . . . . . . . . . . . . . 6.11 Impact of the Human Genome Project on Our Genomic Makeup 6.12 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Molecular Diagnosis in the Post Genomic and Proteomic Era . . . . . . . . . . . . . . . . . . . . 7.1 Prologue . . . . . . . . . . . . . . . . . . . . . . 7.2 The Genomic Era . . . . . . . . . . . . . . . . . 7.3 Concept . . . . . . . . . . . . . . . . . . . . . . 7.4 The Post Genomic Era . . . . . . . . . . . . . . 7.5 Advantages of Combining Multiple Types of Data 7.6 Biomedical Research in the Postgenomic Era . . 7.7 Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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8 Ethics, Patents and Regulations . . . . . . . . . . . . . . . . . . 8.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Concept of Ethical, Legal, and Social Issues . . . . . . . . 8.3 Ethical and Social Issues in Diagnostic Molecular Genetics 8.4 Confronting the Ethical, Legal and Social Issues . . . . . . 8.5 Genes and Disease . . . . . . . . . . . . . . . . . . . . . 8.6 The Existence of Genetic Information . . . . . . . . . . . 8.6.1 Genetic Treatment of Disease . . . . . . . . . . . 8.6.2 Genetic Testing . . . . . . . . . . . . . . . . . . 8.6.3 Discrimination . . . . . . . . . . . . . . . . . . . 8.7 Impact of the Human Genome Project at the Interface Between Patent and FDA Law . . . . . . . . . . . . . . . 8.8 Regulatory Process . . . . . . . . . . . . . . . . . . . . . 8.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9 Polymerase Chain Reaction . . . . . . . . . . . . 9.1 Prologue . . . . . . . . . . . . . . . . . . . 9.2 The Concept of Polymerase Chain Reaction 9.3 PCR Optimisation . . . . . . . . . . . . . . 9.3.1 Magnesium Concentration . . . . . 9.3.2 Buffer Concentration . . . . . . .

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9.3.3 Enzyme Concentration . . . . . . . . . . . . 9.3.4 PCR Primer Design . . . . . . . . . . . . . 9.3.5 Template Quality . . . . . . . . . . . . . . . 9.3.6 Template Quantity . . . . . . . . . . . . . . 9.3.7 Cycling Parameter . . . . . . . . . . . . . . 9.3.8 PCR Enhancers and Additives . . . . . . . . 9.3.9 Nucleic Acid Cross-Contamination . . . . . 9.4 Advances in the PCR Technique . . . . . . . . . . . 9.4.1 RT-PCR . . . . . . . . . . . . . . . . . . . 9.4.2 “Hot Start” PCR . . . . . . . . . . . . . . . 9.4.3 Long Range PCR . . . . . . . . . . . . . . 9.4.4 Inverse PCR . . . . . . . . . . . . . . . . . 9.4.5 Anchored PCR . . . . . . . . . . . . . . . . 9.4.6 Nested Primer PCR . . . . . . . . . . . . . 9.4.7 Colony PCR . . . . . . . . . . . . . . . . . 9.4.8 Quantitative PCR . . . . . . . . . . . . . . 9.4.9 Real-Time PCR . . . . . . . . . . . . . . . 9.4.10 Rapid Amplification of cDNA Ends (RACE) 9.4.11 AFLP . . . . . . . . . . . . . . . . . . . . . 9.4.12 In Situ PCR . . . . . . . . . . . . . . . . . 9.5 Cloning PCR Products . . . . . . . . . . . . . . . . 9.5.1 T-A Cloning Strategy . . . . . . . . . . . . 9.5.2 Incorporation of Restriction Sites in Primers 9.6 PCR as a Diagnostic Tool . . . . . . . . . . . . . . . 9.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

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In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 The Concept of In Situ Hybridization . . . . . . . . . . . . 10.2.1 The Process . . . . . . . . . . . . . . . . . . . . 10.3 Disadavantages of Radioactive Probes . . . . . . . . . . . 10.4 Solving the Problem: Advent of FISH Technique . . . . . 10.4.1 Fluorescence In Situ Hybridization . . . . . . . . 10.4.2 FISH in Action . . . . . . . . . . . . . . . . . . . 10.5 Applications of FISH as a Diagnostic Tool for Research . . 10.5.1 To Analyze the Onset of Specific Gene Expression 10.5.2 Analysis of the Chromosome Structure . . . . . . 10.5.3 Localisation of RNA Transcripts . . . . . . . . . 10.5.4 FISH as a Molecular Cytogenetic Technique to Understand Diseases . . . . . . . . . . . . . . 10.5.5 FISH for Detection of Pathogens . . . . . . . . . 10.6 Recent Advances of In Situ Hybridisation Technology . . . 10.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Types of Immunoassay . . . . . . . . . . . . . . . . 11.4 Recent Advances in the Field of Immunodiagnostics . 11.5 Clinical Applications of Immunoassay . . . . . . . . 11.6 Utilization and Interpretation of Immunological Tests 11.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Phage-Display Libraries as Populations of Replicable, Mutable Chemicals . . . . . . . . . . . . . . . . . . . . . 12.4 Practical Applications of Phage Display . . . . . . . . . . 12.4.1 Target Receptors Used in Affinity Selection . . . . 12.4.2 Epitope Mapping and Mimicking . . . . . . . . . 12.4.3 Identifying New Receptors and Natural Ligands . 12.4.4 Drug Discovery . . . . . . . . . . . . . . . . . . 12.4.5 Epitope Discovery – A New Route to Vaccines and Diagnostics . . . . . . . . . . . . . . . . . . 12.4.6 Selection of DNA-Binding Proteins . . . . . . . . 12.4.7 Landscape Libraries as a Source of New Materials 12.4.8 Phage Display-Combinatorial Chemistry on the Cheap . . . . . . . . . . . . . . . . . . . . . 12.4.9 Cloning Allergens by Phage Display . . . . . . . 12.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Microarray . . . . . . . . . . . . . . . . . . . . . 13.1 Prologue . . . . . . . . . . . . . . . . . . . 13.2 The Concept of Microarray . . . . . . . . . 13.3 Current Challenges of Microarrays . . . . . 13.3.1 Gene Discovery . . . . . . . . . . 13.3.2 Gene Expression Profiling . . . . . 13.3.3 Pharmacogenomics and Microarray 13.3.4 Molecular Diagnostic Research . . 13.4 Conclusion . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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DNA Biosensors . . . . . . . . . . . . . 14.1 Prologue . . . . . . . . . . . . . 14.2 The Concept of DNA Biosensor 14.3 Applications of Biosensors . . . 14.4 Advantages of Biosensors . . . .

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14.5 Development of DNA Hybridization Biosensor . . . . . . . . 14.6 DNA Biosensor for Molecular Detection of Pathogens . . . . . 14.7 Biosensors as Analytical Tools in the Food and Drink Industries 14.8 Potential of Biosensor for Environmental Monitoring . . . . . 14.9 Conclusions & Future Challenges . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Molecular Microbiological Testing . . . . . . . . . . . . . . . . . . 15.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 Advent of Improved Diagnostics . . . . . . . . . . . . . . . . 15.4 Traditional Microbial Typing . . . . . . . . . . . . . . . . . . 15.4.1 Biotyping . . . . . . . . . . . . . . . . . . . . . . . 15.4.2 Antibiograms, Resistograms, and Bacteriocin Typing 15.4.3 Protein Analysis . . . . . . . . . . . . . . . . . . . . 15.4.4 Phage Analysis . . . . . . . . . . . . . . . . . . . . . 15.4.5 Chromatographic Analysis . . . . . . . . . . . . . . . 15.5 Nucleic Acid-Based Typing Systems . . . . . . . . . . . . . . 15.5.1 Plasmid Analysis . . . . . . . . . . . . . . . . . . . . 15.5.2 Restriction Enzyme Pattern . . . . . . . . . . . . . . 15.5.3 Ribotyping . . . . . . . . . . . . . . . . . . . . . . . 15.5.4 Random Amplified Polymorphic DNA (RAPD) . . . 15.5.5 Nucleic Acid Probes . . . . . . . . . . . . . . . . . . 15.5.6 Polymerase Chain Reaction . . . . . . . . . . . . . . 15.6 Current Application of Molecular Diagnostics . . . . . . . . . 15.6.1 Clinical Microbiology . . . . . . . . . . . . . . . . . 15.6.2 Clinical Epidemiology and Infection Control . . . . . 15.7 Promise of Molecular Testing . . . . . . . . . . . . . . . . . . 15.8 Assay Validation-Analytic Sensitivity and Specificity . . . . . 15.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

227 227 228 229 230 230 231 231 231 231 232 232 232 232 232 233 233 234 234 237 238 239 241 241

16

Proteomic Technology . . . . . . . . . . . . . . . . . . 16.1 Prologue . . . . . . . . . . . . . . . . . . . . . . 16.2 Concept . . . . . . . . . . . . . . . . . . . . . . 16.3 Methods to Perform Proteomic Analysis . . . . . 16.4 Functional Proteomics . . . . . . . . . . . . . . 16.5 Implications of the Human Genome Project . . . 16.6 Measurement Using a Proteomic Approach . . . 16.7 Use of Mass Spectrometry in Proteomics . . . . . 16.8 Proteomics: From Basic Research to Diagnostic Application . . . . . . . . . . . . . . . . . . . . 16.9 Role of In Vitro Diagnostics (IVD) in Health Care 16.10 Goals of Proteomics . . . . . . . . . . . . . . . . 16.11 Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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245 245 246 247 249 249 251 252

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Nanomedicine . . . . . . . . . . . . . . . . . . . . . . . . 17.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . 17.2 Concept . . . . . . . . . . . . . . . . . . . . . . . 17.3 Nanotechnology in Medicine . . . . . . . . . . . . 17.3.1 Use of Quantum Dots . . . . . . . . . . . 17.3.2 Cell Targeting . . . . . . . . . . . . . . . 17.3.3 New Nanocarriers for Drug Delivery . . . 17.3.4 Gene Therapy . . . . . . . . . . . . . . . 17.3.5 Molecular Medicine . . . . . . . . . . . . 17.3.6 Construction of Nanostructure Template . 17.3.7 Targeting Drugs to Cells . . . . . . . . . . 17.4 Nanodiagnostics . . . . . . . . . . . . . . . . . . . 17.5 Molecular Electronics . . . . . . . . . . . . . . . . 17.6 Nanotechnology Biosensor for Pathogen Detection 17.7 Nanotechnology Composite Materials for Next Generation Biomedical Applications . . . . . . . . 17.8 A Carbon Nanotube Biosensor . . . . . . . . . . . 17.9 Conclusion & Future Directions . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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261 261 262 264 265 266 267 270 272 273 274 276 278 280

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280 281 282 283

Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . 18.1 Prologue . . . . . . . . . . . . . . . . . . . . . . 18.2 Concept of Biomarkers . . . . . . . . . . . . . . 18.3 Biomarkers on the Horizon . . . . . . . . . . . . 18.3.1 DNA Biomarkers . . . . . . . . . . . . 18.3.2 RNA Biomarkers . . . . . . . . . . . . . 18.3.3 Protein Biomarkers . . . . . . . . . . . 18.4 New Applications for Established Biomarkers . . 18.4.1 Development Pathways . . . . . . . . . 18.4.2 Identification of Biomarkers . . . . . . . 18.4.3 Chronology of Biomarker Development . 18.4.4 Role of Biomarkers in Cancer Detection, Diagnosis, and Prognosis . . . . . . . . 18.4.5 Biomarkers in Cancer Therapy . . . . . 18.4.6 Biomarkers in Early Detection . . . . . . 18.4.7 Biomarkers in Risk Assessment . . . . . 18.5 Organization of a Program in Protein Biomarker Discovery . . . . . . . . . . . . . . . . . . . . . 18.5.1 Cancer Site Teams . . . . . . . . . . . . 18.5.2 Biomarker Mines . . . . . . . . . . . . 18.5.3 Informatics Platform . . . . . . . . . . . 18.5.4 Reagents Core . . . . . . . . . . . . . . 18.5.5 Incorporating Technology Improvements 18.6 Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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287 287 288 289 290 290 291 294 295 295 296

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Diagnosis and Monitoring of Infections . . . . . . . . . . . 19.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . 19.2 Concept of Diagnosis and Monitoring of Infections . 19.2.1 Detection and Identification of Pathogens Without Target Amplification . . . . . . . . 19.2.2 Nucleic Acid Amplification . . . . . . . . . 19.2.3 Detecting Antimicrobial-Drug Resistance . . 19.3 Molecular Diagnostics of Infections . . . . . . . . . 19.3.1 Sexually Transmitted Infections . . . . . . . 19.3.2 Vector Borne Disease . . . . . . . . . . . . 19.3.3 Viral Infections . . . . . . . . . . . . . . . . 19.3.4 Bacterial Infections . . . . . . . . . . . . . 19.3.5 Fungal Infections . . . . . . . . . . . . . . 19.3.6 Practical Applications of Molecular Methods in the Clinical Microbiology Laboratory . . 19.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Diagnosis of Mutation and Genetic Disorders . . . . . . . . . . 20.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3 Factors Regulating a Genetic Disease . . . . . . . . . . . . 20.4 Genetic Testing . . . . . . . . . . . . . . . . . . . . . . . 20.4.1 Cytogenetic Testing . . . . . . . . . . . . . . . . 20.4.2 Biochemical Testing . . . . . . . . . . . . . . . . 20.4.3 Molecular Testing . . . . . . . . . . . . . . . . . 20.5 Current Status of Molecular Diagnosis of Some Common Genetic Diseases . . . . . . . . . . . . . . . . . 20.5.1 Cystic Fibrosis . . . . . . . . . . . . . . . . . . . 20.5.2 Duchenne Muscular Dystrophy . . . . . . . . . . 20.5.3 Haemophilia A . . . . . . . . . . . . . . . . . . . 20.5.4 Haemophilia B . . . . . . . . . . . . . . . . . . . 20.5.5 Phenylketonuria . . . . . . . . . . . . . . . . . . 20.5.6 β Thalassaemia . . . . . . . . . . . . . . . . . . . 20.5.7 Wilson’s Disease (Hepatolenticular Degeneration) 20.5.8 α Antitrypsin Deficiency . . . . . . . . . . . . . . 20.5.9 Familial Hypercholesterolaemia and Other Lipoprotein Disorders . . . . . . . . . . . . . . . 20.5.10 Huntington’s Disease Gene . . . . . . . . . . . . 20.5.11 Multiple Endocrine Neoplasia . . . . . . . . . . . 20.5.12 Factor V (Leiden) Mutation . . . . . . . . . . . . 20.5.13 Hemochromatosis Gene, HFE . . . . . . . . . . . 20.5.14 Colon Cancer Gene, APC . . . . . . . . . . . . . 20.6 Microarray Analysis for Detection of Complex Pattern of Genes . . . . . . . . . . . . . . . . . . . . . . .

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20.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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21

Diagnosis of Complex Diseases . . . . . . . . . . . . . . . . . 21.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Background Information . . . . . . . . . . . . . . . . . 21.4 Detecting New Metabolic Disease Pathways . . . . . . . 21.5 Phenotypic Variations in Simplex Diseases . . . . . . . . 21.6 The Genetic Background of Complex Disorders . . . . . 21.7 Dissecting Interactions Between Genes and Environment 21.7.1 Alzheimer Disease . . . . . . . . . . . . . . . . 21.7.2 Cancer . . . . . . . . . . . . . . . . . . . . . . 21.7.3 Multiple Sclerosis . . . . . . . . . . . . . . . . 21.7.4 Diabetes . . . . . . . . . . . . . . . . . . . . . 21.7.5 Hemophilia . . . . . . . . . . . . . . . . . . . . 21.7.6 Obesity . . . . . . . . . . . . . . . . . . . . . . 21.7.7 Schizophrenia . . . . . . . . . . . . . . . . . . 21.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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347 347 348 349 350 350 351 351 353 356 360 363 364 367 370 374 375

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Biochips . . . . . . . . . . . . . . . . . . . . . 22.1 Prologue . . . . . . . . . . . . . . . . . 22.2 Concept . . . . . . . . . . . . . . . . . 22.3 Design of Biochip . . . . . . . . . . . . 22.4 Applications of Biochips . . . . . . . . 22.4.1 Biochip and Clinical Medicine 22.4.2 Biochips and Biosensors . . . 22.4.3 Biochip and Pharmacogenetics 22.4.4 Biochip and Biowarfare . . . . 22.4.5 Biochip and Diagnostics . . . . 22.4.6 Biochip and Cancer . . . . . . 22.4.7 Biochip Department of Defense 22.5 Biochip Market . . . . . . . . . . . . . 22.6 Future of Biochips . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Personalised Medicine . . . . . . . . . . . . . . . . . . . . . . . . . 23.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 Practice of Medicine in the Twenty-First Century . . . . . . . 23.4 Role of Personalized Medicine in Drug Discovery . . . . . . . 23.5 Molecular Diagnosis Will Determine Prognosis and Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.6 Personalized Medicine and Genetic Markers . . . . . . . . . . 23.7 Challenges of Realizing the Promise of Personalized Medicine

393 393 394 396 396

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23.8 Personalised Medicine and Pharmacogenomics . . . . . 23.9 Personalized Medicine and Diseases . . . . . . . . . . . 23.10 Personalized Medicine and Diagnostics Industry . . . . . 23.11 Impact of the US Patent System on the Promise of Personalized Medicine . . . . . . . . . . . . . . . . . 23.12 Ethical Legal and Social Issues of Personalised Medicine 23.13 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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403 406 406

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24

Biopharmaceutical Industry and Health Care . . . 24.1 Prologue . . . . . . . . . . . . . . . . . . . . 24.2 Concept . . . . . . . . . . . . . . . . . . . . 24.3 Biopharmaceutical Research . . . . . . . . . 24.4 Opportunities in Healthcare . . . . . . . . . . 24.5 Molecular Diagnostics and Health Care . . . 24.6 Global Context . . . . . . . . . . . . . . . . 24.7 Emerging Biopharmaceuticals . . . . . . . . 24.8 Challenges of the Biopharmaceutical Industry References . . . . . . . . . . . . . . . . . . . . . . .

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Forensic Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25.3 Forensic Medicine and DNA Fingerprinting . . . . . . . . . . 25.4 Applications of DNA Methylation Markers in Forensic Medicine 25.5 Forensic Medicine and Anthropometry . . . . . . . . . . . . . 25.6 Clinical Forensic Medicine . . . . . . . . . . . . . . . . . . . 25.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

425 425 426 428 431 431 432 433 433

26

Pharmacogenomics . . . . . . . . . . . . . . . . . . . . 26.1 Prologue . . . . . . . . . . . . . . . . . . . . . . 26.2 Concept . . . . . . . . . . . . . . . . . . . . . . 26.3 Predicting Drug Response on Gene Variation . . 26.4 Drug Development and Testing Benefit from Pharmacogenomics . . . . . . . . . . . . . 26.5 Applications and Benefits of Pharmacogenomics . 26.5.1 Better, Safer Drugs the First Time . . . . 26.5.2 More Accurate Methods of Determining Appropriate Drug Dosages . . . . . . . 26.6 Benefits of Pharmacogenomic Testing . . . . . . 26.6.1 Patient’s Ability to Metabolize Drugs . . 26.6.2 Age-Related Genetic Variations . . . . . 26.7 Recent Reports of Pharmacogenomics Use . . . . 26.8 Interpreting Pharmacogenomic Tests . . . . . . . 26.9 Barriers to Pharmacogenomics Progress . . . . .

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The Promise of Pharmacogenomics . . . . . . . . . . . . . . Impact of Drug Discovery, Development, and Marketing Affected by Pharmacogenomics . . . . . . . 26.12 Pharmacogenomics Initiatives . . . . . . . . . . . . . . . . 26.13 Pharmacogenomics and the Pharmaceutical Industry . . . . . 26.14 Applied Pharmacogenetics . . . . . . . . . . . . . . . . . . 26.15 Ethical Concerns . . . . . . . . . . . . . . . . . . . . . . . 26.16 Molecular Diagnostic Methods for Optimizing Drug Therapy 26.17 Challenges for the Future . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Gene Technology in Forensic Sciences . . . . . . . . . . . . . . . . 27.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27.3 DNA Typing and Genetic Markers . . . . . . . . . . . . . . . 27.4 DNA Based Methods for Identification of Individuals . . . . . 27.4.1 Non-PCR Based Methods . . . . . . . . . . . . . . . 27.4.2 PCR Based Methods . . . . . . . . . . . . . . . . . . 27.5 Ethical, Legal, and Social Concerns About DNA Databanking 27.6 Future of DNA Typing Systems . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

455 455 456 456 459 459 460 463 463 464

28

Food Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . 28.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . . . . 28.3 Tools of Molecular Diagnostics to Assess Food Quality . . 28.4 Food Allergy . . . . . . . . . . . . . . . . . . . . . . . . 28.5 Food-Borne Disease . . . . . . . . . . . . . . . . . . . . . 28.6 Commercial Rapid Food-Safety Diagnostics . . . . . . . . 28.7 Methods for Analysis of Food . . . . . . . . . . . . . . . 28.7.1 Rapid Methods . . . . . . . . . . . . . . . . . . . 28.8 Future Technologies . . . . . . . . . . . . . . . . . . . . . 28.8.1 Microarrays . . . . . . . . . . . . . . . . . . . . 28.8.2 Sensor Technology . . . . . . . . . . . . . . . . . 28.8.3 Flow Cytometry . . . . . . . . . . . . . . . . . . 28.8.4 Bacteriophage-Based Techniques . . . . . . . . . 28.8.5 Riboprinting and Pulse-Field Gel Electrophoresis 28.8.6 Applications and Limitations of Rapid Methods . 28.9 Business Outlook of Food Diagnostics . . . . . . . . . . . 28.10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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467 467 468 469 470 470 471 472 473 476 476 477 478 478 479 479 479 480 481

29

Rapid Diagnostic Methods for Biowarfare . 29.1 Prologue . . . . . . . . . . . . . . . . 29.2 Concept . . . . . . . . . . . . . . . . 29.3 Characteristics of Biological Weapons

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29.3.1 Anthrax . . . . . . . . . . . . . . . . 29.3.2 Plague . . . . . . . . . . . . . . . . . 29.3.3 Tularemia . . . . . . . . . . . . . . . 29.3.4 Melioidosis . . . . . . . . . . . . . . . 29.3.5 Viral Hemorrhagic Fevers . . . . . . . 29.3.6 Other Viral Fevers . . . . . . . . . . . 29.3.7 Trichothecene Mycotoxins . . . . . . . 29.3.8 Aflatoxin . . . . . . . . . . . . . . . . 29.4 Biodefense . . . . . . . . . . . . . . . . . . . . 29.4.1 Viral Agents – Poxviridae . . . . . . . 29.4.2 Botulinal Toxins . . . . . . . . . . . . 29.4.3 Mycotoxins . . . . . . . . . . . . . . 29.5 Combating Detection of Biowarfare Agents . . 29.5.1 Prophylatic & Therapeutic Approaches 29.5.2 Detection Methods . . . . . . . . . . . 29.6 Impact of Biological Weapons . . . . . . . . . 29.7 Tools for Self Defence Against Bioweapons . . 29.8 Genetic Engineering and Biological Warfare . . 29.9 Impact of Genomics and Genetic Technology . 29.10 Conclusion . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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487 487 487 488 488 488 488 489 489 490 490 491 491 491 495 495 496 497 499 500 500

Segments of Molecular Diagnostics – Market Place . . . . 30.1 Prologue . . . . . . . . . . . . . . . . . . . . . . . . 30.2 Concept . . . . . . . . . . . . . . . . . . . . . . . . 30.3 Market Drivers . . . . . . . . . . . . . . . . . . . . 30.4 Trends in Infectious Diseases Testing Market . . . . 30.5 Trends in Cancer Diagnostic Testing World Markets . 30.6 Trends in Cardiac Marker Diagnostic Testing Markets 30.7 Point of Care Diagnostic Testing World Markets . . . 30.8 Next Generation Molecular Diagnostics . . . . . . . 30.9 The Rise of Companion Diagnostics . . . . . . . . . 30.10 Market Considerations and Forecasts . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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503 504 504 506 507 507 508 508 509 510 510 513

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

515

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Chapter 1

Introduction to Molecular Diagnostics

Abstract Molecular diagnostics has become a growing part of the clinical laboratory. It includes all tests and methods to identify a disease and understand the predisposition for a disease analyzing DNA or RNA of an organism. Rapid advances in molecular diagnostics enable basic research and results in practical diagnostic tests. The basic application is to determine changes in sequence or expression levels in crucial genes involved in disease. The use of molecular diagnostics, such as pre-implantation diagnostics or predictive genetic testing, still has technical problems as well as novel, and to date unclear, social, ethical and legal implications. The scope of molecular diagnostics in molecular medicine could be expanded well beyond current nucleic acid testing. It plays an important role in practice of medicine, public health, pharmaceutical industry, forensics and biological warfare and drug discovery. The molecular diagnostic marketplace offers a growth opportunity given the interest in utilizing molecular tools to precisely target therapeutics. Keywords Clinical diagnostics · Molecular biology · Health care · Molecular diagnostics · Genetics · Diagnostic medicine · Prognostic · Molecular diagnostic industry · Clinical testing · Therapeutics · PCR · Microarray

1.1 Prologue The past few years has seen enormous progress in molecular genetics. Techniques such as Southern blotting, Northern blotting, polymerase chain reaction, nucleotide sequencing, chromosome walking and genetic transfer allow the specific isolation, characterization and modification of genetic information (Boerman et al., 2001). Molecular studies allow the laboratory to become predictive. Now statements can be made about events that occur to a patient in the future. This new technology returns results that give an indication that the patient may be at risk for the disease long before it becomes symptomatic disease. Molecular pathology is in a state of rapid evolution featuring continuous technology development and new clinical opportunities for drug selection predicting efficiency, toxicity and monitoring disease outcome. Thus major advances are being M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_1,

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made in the science of genetics, resulting in increased use of molecular technology in clinical laboratory.

1.2 Clinical Diagnostics Entering a New Phase Biotechnology, in all its forms, is the fastest-growing discipline in the modern laboratory, whether be it clinical, research or forensic (Jain, 2002). The volume of research from the past is translating to a growing list of new diagnostic tests for the patient oriented clinical laboratory. Optimists have characterized these developments as “mankind about to determine its destiny,” whereas others are afraid that scientists are about to “start playing God.” Molecular biology has revolutionized biological and biomedical research and has become an indispensable tool in clinical diagnostics (Fig. 1.1). It has developed more than any other science in the last 10 years. Until this time, the laboratory had been descriptive in nature. It could measure events that were currently going on by evaluating the chemistry, hematology or anatomical pathology. Major advances are being made in the science of genetics, resulting in the increased use of molecular technology in the clinical laboratory. A wide variety of drugs in late preclinical and clinical development are being targeted to disease specific gene and protein defects that will require co-approval of diagnostics and therapeutics products by regulating agencies. An increasingly educated public will demand more information about their predisposition for serious diseases, how these potential illnesses can be detected in an early stage, when they can be arrested or cured with new therapies custom-designed for their individual clinical status. To respond to this demand, major pharmaceutical companies will form partnerships with diagnostics companies or develop their own in-house capabilities that will permit efficient production of more effective and less toxic integrated personalized medicine drug and test products. For clinical laboratories and pathologists, this integration of diagnostics and therapeutics represents a major new opportunity to emerge as leaders of the new medicine, guiding the selection, dosage, route of administration, and multidrug combinations and producing increased efficacy and reduced toxicity of pharmaceutical products.

Fig. 1.1 Applications of molecular diagnostics in the field of clinical diagnostics

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Concept of Molecular Diagnostics

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The clinical laboratory has a long history of developing, managing, and providing assays and test results for diagnosing disease. A good example of this is the development of immunoassays and the enhancements made to this technology with the introduction of monoclonal antibodies. Clinical molecular genetics is now part of the mainstream of medical care in the United States (Amnos and Patnaik, 2002). All commercial clinical reference laboratories now have a molecular genetic diagnostic unit, many of which are in contractual agreement with third party payers to provide services. Gene discovery provides valuable insight into the mechanisms of disease processes and gene-based markers will enable clinicians to study disease predisposition, as well as improved methods for diagnoses, prognosis, and monitoring of therapy. The broad range of mutation spectrum and type performed in the clinical laboratory requires the use of multiple technologies rather than a single typing platform. Platform choice depends on such diverse factors as local expertise, test volume, economies of scale, R&D budget, and royalties. Test validation is a major hurdle and positive control samples are often not readily available. Oversight and the regulatory environment for clinical molecular genetics laboratories in the United States are evolving rapidly. Several government agencies and private organizations are currently involved in revision of specific laboratory standards, including the Secretary’s Advisory Committee on Genetic Testing (SACGT), Food and Drug Administration (FDA), Center for Disease Control (CDC), College of American Pathologists (CAP), American College of Medical Genetics (ACMG). Over the past 50 years, the clinical laboratory has evolved into a complex, technology-driven enterprise with the principal tasks of diagnosing and screening for disease, monitoring health and therapeutic response, and gauging deviations from normal physiology in humans and animals. Advances in diagnostic medicine, on the other hand, have come through the application of science and technology as a result of a coordinated effort among academia, industry, government, and private institutions. We are now entering the era of Molecular Diagnostics and Pathology, which is bringing forth the newest and most powerful science and technology available for the modern-day practice of diagnostic laboratory medicine. Among the numerous important areas to consider with molecular diagnostics are the emerging issues concerning the development of genetic assays and their use for testing individual patient responses or suitability for pharmaceutical drugs. The rapidly growing area of molecular diagnostics is ideally suited to clinical laboratories (Hall et al., 2003).

1.3 Concept of Molecular Diagnostics Molecular diagnosis is the detection of pathogenic mutations in DNA and RNA samples to aid in detection, diagnosis, subclassification, prognosis, and monitoring response to therapy. It covers current molecular biological techniques used to identify the underlying molecular defects in inherited disease. Principles underlying nucleic acid based diagnosis originate from localization, identification, and characterization of genes responsible for human disease. It also determines how

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these genes and proteins are interacting in a cell. It focuses upon patterns, gene and protein activity patterns in different types of cells. Molecular diagnostics uncovers these sets of changes and captures this information as expression patterns. Also called “molecular signatures,” these expression patterns are improving the clinicians’ ability to diagnose diseases. Molecular Diagnostics is constantly translating new discoveries and novel technologies into useful clinical tests that provide a molecular fingerprint of tumors and they are predictive of the response to specific therapies. Molecular diagnostic technologies offer the potential for moving from diagnostics to prognostics. More than 80% of molecular tests performed today address infectious disease detection and management. The test menu for infectious diseases includes human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), cytomegalovirus (CMV), Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycobacterium tuberculosis. Other molecular tests are applied in forensic medicine, paternity testing, tissue typing, oncology, and food and beverage testing. The molecular diagnostic testing can be performed on very small amounts of tissue obtained from small biopsies and fine-needle aspirates. Testing can even be performed on archival material (material that has been collected in the past and fixed in formalin). It is usually not necessary to take extra tissue for these tests, since most of them can be performed on tissue that is left over after routine diagnostic analysis.

1.4 Molecular Diagnostic Technology and Health Care Industries Advancing knowledge and technologies are pushing the diagnostics industry to the front, and the molecular diagnostics industry in particular is emerging as a powerful health care player with tremendous potential. Molecular diagnostics is the fastest growing segment of clinical testing today, comprising 3–4 times the growth of the core business, depending on the country. The molecular diagnostic market is expected to grow 15–17% annually in the short term. Anticipation of such steady growth is based on expectations that maturation of the technology, the development of diagnostic applications, and expansion of the market into developing countries will lead to increased acceptance and market penetration of the tests. Continuing simplification of the technology and instrumentation used in molecular diagnostic tests holds the key to this phase of development. In the US, this sector is predicted to expand to 30% of the total service diagnostics revenues by 2015. Traditionally, it has only included testing for infectious disease (such as HIV and HPV); genetics (such as cystic fibrosis, and BRCA1 and 2); and, to a lesser extent, oncology (such as leukemia and lymphoma molecular classification). However, trends are emerging based on new molecular tests for the diagnosis and management of several diseases beyond cancer, infectious disease and genetics. This includes testing for immunity, metabolism, the central nervous system and cardiovascular function. In

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Molecular Diagnostic Technology and Health Care Industries

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addition, new technologies which analyze multiple genes (10–10,000), single cells or new metabolites are now available, providing rapid information to better classify or manage disease. Recent progress in the development of molecular diagnostics in medicine has been rapid and the hope has been expressed by some people that it will soon be possible to have a detailed “genetic readout” to assist in the diagnosis of treatment of a variety of diseases (Hall et al., 2003). The genomic discovery will fuel the diagnostic marketplace because every gene-based therapy might need an accompanying (molecular) diagnostic test. At approximately 30,000 genes for the human genome and with an estimated diagnostic significance of about 5%, the commercialization of 1,500 gene-based tests can be expected. The way is paved by infectious disease testing and blood banking applications, but pharmacogenetic, predisposition diagnostics and molecular cancer diagnostics applications will follow soon and post strong numbers in the years to come. Cancer for instance, represents a set of diseases with vast unmet clinical need for improved diagnostics and therapeutics. Cancer in the developed western countries with prevalence between 1.0 and 1.5% depicts a large market with a high potential for R&D and fields like therapy specific diagnostics (theranostics). The function of such technology is to determine changes in sequence or expression levels in crucial genes involved in cancer. Molecular diagnostics are seen as being a major breakthrough in medical science. Rapid progress in this field is expected to fundamentally change health care (Jain, 2002). While the technology itself has broad applications to such areas as agricultural bioterrorism and environmental science, medical and medical research applications have provided the primary focus. As a result, great things have been predicted for the development and use of new therapeutics based on molecular testing approaches. Gene-based molecular diagnostics is changing the practice of medicine and will continue to do so for the foreseeable future. The primary molecular diagnostics approaches are based on two major database sciences (genomics and proteomics) and their relationship to disease and metabolic processes (functional genomics and functional proteomics). The genome or proteome that forms the basis for these tests is very often of human origins, but may also be from a pathogen. As a result, the breadth of application for molecular diagnostics is virtually unlimited. With all this opportunity, however, progress has been slow in the development, introduction and commercialization of molecular diagnostics. This has been related to the difficulty and expense of performing such assays, and the utility of the test procedures in the absence of substantive therapeutic options based on the additional data provided by these assays. The main objectives of this analysis are: (1) To identify viable technology drivers through a comprehensive look at platform technologies for molecular diagnostics, including probe-based nucleic acid assays, microarrays and sequencing;

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(2) To obtain a complete understanding of the chief molecular diagnostics tests i.e., predictive, screening, prognostic, monitoring, pharmacogenomic and theranostic from their basic principles to their applications; (3) To discover feasible market opportunities by identifying high-growth applications in different clinical diagnostic areas and by focusing on expanding markets, such as communicable diseases, cardiology and oncology; and (4) To focus on global industry development through an in-depth analysis of the major world markets for molecular diagnostics, including growth forecasts. The molecular diagnostic industry is characterized by a diverse, constantly changing technology base that continuously produces new opportunities and applications which has become a growing part of the clinical laboratory in recent years. The trend will not only continue but will show significant growth in the future. Similarly, a growing understanding of the molecular basis of cancer and other chronic diseases is opening up new realms of medicine to the possibilities of molecular diagnostic testing. Molecular signatures can also be important for the detection of minimal residual or recurrent disease and can help distinguish a metastasis from a second primary tumor. The tests are highly accurate and sensitive. When interpreted within the appropriate clinical situation and combined with other diagnostic procedures, these tests can help health care providers diagnose lymphomas and other tumors earlier so that appropriate treatment can be started. Genetic screening tests, despite some restrictions are a promising area for future expansion of in vitro diagnostic market. Molecular diagnostics is being combined with therapeutics and forms an important component of integrated healthcare. Molecular diagnostic technologies are also involved in development of personalized medicine based on pharmacogenetics and pharmacogenomics. Currently, there has been a considerable interest in developing rapid diagnostic methods for point-of-care and biowarfare agents such as anthrax. To date, the technical elements of molecular therapy have not yet fulfilled their expectations. In the broad spectrum of obstetrics and gynecology, new molecular discoveries are influenced not only by technical but also by socioeconomic and political considerations. These include, for example, free access to genetic testing, patents for genes and the financial monopoly over molecular medication (Beckmann et al., 2003).

1.5 Molecular Diagnostic Approaches Molecular diagnostics focuses primarily on nucleic acids. Rapid advances in molecular diagnostics both enable basic research and result in practical diagnostic tests (Dennis and Wittwer, 2009). These types of tests include the analysis of DNA, RNA (nucleic acids) micro RNAs, and complex proteomic or metabolomic pattern array based tests. In the foreseeable future, proteomic/metabolomic tests may form their own sub-category (Fig. 1.2). However, they are presently included among the general group of molecular diagnostics.

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Molecular Diagnostic Approaches

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Fig. 1.2 The diagnostic business includes all service laboratories and in vitro diagnostics

1.5.1 Nucleic Acid Test Systems The field of molecular diagnostics is represented primarily by the nucleic acid test (NAT), which is used to detect genomic DNA or RNA sequences and to characterize genomic aberration or sequence polymorphisms. For the past 20 years, NATs have been mainly hybridization-based assays, with detection a function of label molecules coupled to the probe sequence. Some commercial NATs are based on direct hybridization detection of ribosomal RNA. The high sensitivity of the assay is attributable partly to the fact that there are thousands of ribosomal RNA molecules per cell. To be able to detect less-abundant RNA or DNA molecules at increased sensitivity usually requires some form of amplification to generate a detectable signal. Amplification strategies vary, but techniques can be categorized as target, probe, or signal amplifications. Target amplification employs various sequences of enzymatic reactions to amplify the targeted nucleic acid fragment, that is, to produce multiple copies of this target sequence. Most notable of the target amplification tests is polymerase chain reaction (PCR), developed by Roche Diagnostics Corp. (Indianapolis). Also worth mentioning are strand-displacement amplification (SDA) from BD (Franklin Lakes, NJ), transcription-mediated amplification (TMA) from Gen-Probe Inc. (San Diego), and nucleic acid sequence-based amplification (NASBA) from Organon Teknika Corp. (Durham, NC). All of the hybridizationbased assays may use chromogenic, fluorescent, or optical reporter labels to produce the measurable light units detected with an optical readout device. Today, polymerase chain reaction (PCR) is the gold standard for molecular assays, owing to its intrinsic versatility and flexibility. Advances in PCR, multiplexing, sequencing, and other technologies are propelling both new and old companies forward with novel capabilities. Roche’s PCR-based product lines, which have application strictly in the infectious diseases area, have captured 47% of the molecular diagnostics market.

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This circumstance will not change until molecular assays for cancer and heritable diseases become part of the routine test menu. The objective of molecular test manufacturers is to develop homogeneous closedtube molecular test systems that are suited for diagnostic applications. A number of labeling technologies for homogeneous molecular assays have been developed that employ various configurations of probe and fluorescence label, and incorporate different versions of fluorescence resonance energy transfer (FRET). In a FRET-based assay, a fluorescent-tagged molecular probe is excited by a laser. The fluorophore responds by emitting energy that is transferred to a second, quenching, fluorophore. If these two fluorophore molecules are very close to each other, the fluorescence is quenched. The desire to find better ways to design and manufacture these labels and probes, so as to achieve better performance and a lower cost per test, drives the development of new labeling methods in this area. Now, homogeneous-label methods are widely used by molecular laboratories in in-house PCR tests for infectious diseases, heritable diseases, and cancer. Several IVD manufacturers are incorporating these methods in their own molecular assays. Although homogeneous assays often provide the best sensitivity in detection, the limited number of different fluorophores available for simultaneous detection of multiple analytes from a single sample makes this assay platform less attractive for use in multiplex assays.

1.5.2 Gene-Based Diagnostics Molecular biology has revolutionized biological and biomedical research and has become an indispensable tool in clinical diagnostics. Gene expression profiling, and modifications at the protein levels, will be critical to cancer classification and staging. Lately, demand for multiplexing platforms that perform parallel nucleic acid or protein analyses in diagnostic applications has been steadily increasing. There has been a dramatic rise in the use of DNA microarrays for medical research over the last few years. Although generally perceived as a method for testing thousands of gene fragments with low sensitivity and reproducibility, the GeneChip from Affymetrix Inc. (Santa Clara, CA) is an example and advanced microarray platforms are being devised for diagnostic ends. New platforms, like the Flow-Thru Chip developed by MetriGenix Inc. (Gaithersburg, MD), promise high assay performance, amenability to automation, and the flexibility to handle low- to high-throughput diagnostic applications. These microarrays will be targeting tens to hundreds of analytes rather than thousands. More important, they will combine bioinformatic and microfluidic technologies with the microarray in order to miniaturize and simplify the tests. These new tools allow us to analyze thousands of genes simultaneously leading to a better understanding of gene function and dysfunction.

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Molecular Diagnostic Approaches

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The first commercially available microarray, the AmpliOnc I from Abbott, was developed from a subsidiary’s Vysis GenoSensor microarray system. It is designed to detect abnormal increases in gene copies for 58 different genes. A number of biochip makers are pursuing collaborative arrangements with IVD manufacturers and drug manufacturers to exploit the possibility of their microarray technologies being used in clinical product development. In addition to the microarray platform, bead-based technologies are also gaining recognition for their potential utility in clinical multiplex testing. Each bead incorporates a different label dye and thus has a different spectral address. One can be distinguished from another by its unique wavelength. If each bead is associated with only one specific analyte, then multiplex assays of nucleic acid or protein can be performed. The flexibility of the multiplex format of bead-based assays is the biggest advantage these assays have over the microarray platform. The LabMAP system from Luminex Corp. (Austin, TX) is an example. However, this system carries a disadvantage in that it is limited to fewer than 100 elements per test. Other technologies such as mass spectroscopy and denaturing high-pressure liquid chromatography have also recently been making their way into the molecular diagnostics field. The involvement of thousands and millions of genetic markers in specific diseases and physiological pathways has just begun to be biologically validated. Development of biological assay content in this field will take much longer than technology development. But anticipation is high that, with the large-scale genediscovery efforts being undertaken by many government and private institutions, molecular diagnostics will come to maturity in the next 5–10 years. The market potential for oncological and genetic-disease molecular testing is smaller in the near term than that for infectious-disease testing. For the foreseeable future, in-house molecular tests, offered as analyte specific reagents (ASRS), will remain an integral part of the testing in this field. Only an estimated 25% of the 4,000 known hereditary diseases can be analyzed by means of molecular testing techniques today. Additional factors, including technical, patent, and ethical issues, may further restrict the development and market penetration of genetic diagnostic tests. In this molecular medicine era, it has become widely accepted that almost every disease process results from the interaction of the environment and individual lifestyle with a specific genetic predisposition. There will be no more “one drug fits all,” and pharmaceutical medicines will be prescribed to targeted patient populations case by case on the basis of the individual’s genomic profile. Furthermore, with early detection and intervention available, most of the historically life-threatening diseases will become merely chronic disease conditions that require long-term management. Pharmacogenomic testing for patient drug susceptibility, dose response and drug resistance thus holds exciting potential for molecular diagnostics in years to come. According to a presentation at a recent industry conference, 370 biopharmaceutical drugs are in late stages of clinical development. Management of several medical conditions, including diabetes, asthma, autoimmune disease, and cardiac disease, will greatly benefit from these gene-based diagnostics and therapeutic interventions.

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1.6 Conclusion Molecular diagnostic technologies will play an important role in practice of medicine, public health, pharmaceutical industry, forensics and biological warfare in the twenty-first century. This includes several polymerase chain reactions (PCR) based technologies, fluorescence in situ hybridization (FISH), peptide nucleic acids (PNA), electrochemical detection of DNA, biochips, nanotechnology (Jain, 2003) and proteomic technologies. Molecular diagnostics has evolved rapidly during the past decade and has an impact on the practice of medicine as well as many other applications including drug discovery. Advances in biotechnology have been incorporated into molecular diagnostics. There has been a distinct trend in miniaturization with development of biochips and micro fluidics. This trend has continued with the development of nanotechnology. Nanotechnologies are now being applied to molecular diagnostics to refine and extend the limits of detection (Jain, 2003). The molecular diagnostics offers a growth opportunity in utilizing molecular tools to precisely target therapeutics. The impact of single nucleotide polymorphisms (SNPs) and other molecular diagnostic markers is now being felt. This will grow as more molecular markers are validated and launched for their diagnostic and prognostic value. Ideally, therapy will be based on this information, aiming at the most effective mechanism based treatment with the least toxicity (Zlotogorski et al., 2002). Molecular diagnostics, in the future, will be expected to provide gene profile based personalized therapeutic approaches.

References Amos, J., Patnaik, M., 2002, Commercial molecular diagnostics in the U.S.: The Human Genome Project to the clinical laboratory. Hum Mutat 19:324–333. Beckmann, M.W., Strick, R., Strissel, P.L., Fasching, P.A., Oppelt, P., Pöhls, U.D., Malur, S.U., Ackermann, S., 2003, Aspects of molecular diagnostics and therapy in obstetrics and gynecology. Expert Rev Mol Diagn 3:279–287. Boerman, O.C., Oyen, W.J.C., Corstens, F.H.M., 2001, Progress in gene therapy: seeing is believing. J Nucl Med 42(8):1235–1237. Dennis Lo, Y.M., Wittwer, C.T., 2009, Molecular diagnostics: at the cutting edge of translational research. Clin Chem 55:601. Hall, A.G., Coulthard, S.A., Irving, J.A., 2003, Molecular diagnostics: a healthcare perspective. Expert Rev Mol Diagn 3:13–16. Jain, K.K., 2002, Current trends in molecular diagnostics. Med Device Technol 13:14–18. Jain, K.K., 2003, Nanodiagnostics: application of nanotechnology in molecular diagnostics. Expert Rev Mol Diagn 3:153–161. Zlotogorski, A., Panteleyev, A.A., Aita, V.M., Christiano, A.M., 2002, Clinical and molecular diagnostic criteria of congenital atrichia with papular lesions. J Invest Dermatol 118:887–890.

Chapter 2

Omics Technology

Abstract Omics has become the new mantra in molecular research. “Omics” technologies include genomics, transcriptomics, proteomics and metabolomics. Genomics had revealed the static sequences of genes and proteins and focus has now been shifted to their dynamic functions and interactions. Transcriptomics, proteomics and metabolomics reveal the biological function of the gene product. The “-omic-” technologies are high-throughput technologies and they increase substantially the number of proteins/genes that can be detected simultaneously to relate complex mixtures to complex effects in the form of gene/protein expression profiles. The primary aim of omic technologies is the nontargeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample. The powerful “omics” technologies have opened new avenues towards biomarker discovery, identification of signaling molecules associated with cell growth, cell death, cellular metabolism and early detection of cancer. Omics will not only have an impact on our understanding of biological processes, but the prospect of more accurately diagnosing and treating disease will soon become a reality. Keywords Omics · Genome · Proteomics · Transcriptomics · Metabolomics · Systems biology · High throughput technology · Genomics · Human genome project · Gene sequence · Gene transcript · Herceptin · Gleevec · Molecular signature · Expression profile · Expression signature · Computational tools · Oncology · Proteome · Biomarkers · Yeast two hybrid analysis · Clinical diagnostics · Protein–protein interaction · Mass spectroscopy · Protein microarray · Metabolome · Metabolic profiling · Toxicogenomics · Nutrigenomics

2.1 Prologue Over the last decade, we have witnessed a fundamental change in how biomedical research is carried out and we can now assess the impact of the Human Genome Project on drug discovery and development (Bilello, 2005). With the completion of a rough draft of the human genome, many researchers are looking at how genes and proteins interact to form other proteins. The human genome sequence has revealed that sequence variations are very common. Genetic approaches have already linked M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_2,

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a large number of genetic variations (polymorphisms) with human diseases and adverse reactions from exposure to drugs or toxicants, suggesting sensitivity of genome to drugs and environmental agents, disease susceptibilities, and therapeutic responses. The application of the “-omic-” technologies may lead to a change of paradigms and open the new vistas in medicine.

2.2 Concept of Omics New technologies that permit simultaneous monitoring of many hundreds or thousands of macro- and small molecules promise to allow functional monitoring of multiple (or perhaps all) key cellular pathways simultaneously. The new “global” methods of measuring families of cellular molecules, such as RNA, proteins, and intermediary metabolites have been termed “-omic” technologies, based on their ability to characterize all, or most, members of a family of molecules in a single analysis (Fig. 2.1). With these new tools, we can now obtain complete assessments of the functional activity of biochemical pathways, and of the structural genetic (sequence) differences among individuals and species, that were previously unattainable. The terms “Ome” and “Omics” are derivations of the suffix -ome, which has been appended to a variety of previously existing biological terms to create names for fields of endeavor like genome, proteome, transcriptome and metabolome that are either speculative or have some tangible meaning in particular contexts. Omics has become the new mantra of molecular biology. “Omic” technologies include genomics, transcriptomics (gene expression profiling), proteomics and metabolomics. The recent availability of masses of omic data is responsible for the major growth spurt of systems biology. Pharmaceutical companies and others need to make sense out of all this omic information in order to take the next step in overcoming their innovation deficits. Systems biology provides the methods, computational capabilities, and inter-disciplinary expertise to facilitate this jump. The technology platform of genomics, transcriptomics, proteomics and metabolomics provide high-throughput technologies. They increase substantially the number of proteins/genes that can be detected simultaneously and have the potential to relate complex mixtures to complex effects in the form of gene/protein expression profiles.

Fig. 2.1 The Central Dogma and the interacting “ome” includes the study of genome, proteome, transcriptome and metabolome

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Genome and Genomics

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Commercial applications are emerging at an accelerating pace as pharmaceutical and biotechnology research organizations start to combine various forms of omic data into more comprehensive computer models, and bioinformatics companies increasingly turn their attention toward offering systems biology solutions to drug developers and diagnostics companies.

2.3 Genome and Genomics In biology the genome of an organism is its whole hereditary information and is encoded in the DNA (or, for some viruses, RNA). This includes both the genes and the non-coding sequences of the DNA (Min et al., 1972). More precisely, the genome of an organism is a complete DNA sequence of one set of chromosomes; for example, one of the two sets that a diploid individual carries in every somatic cell. The term genome can be applied specifically to mean the complete set of nuclear DNA (i.e., the “nuclear genome”) but can also be applied to organelles that contain their own DNA, as with the mitochondrial genome or the chloroplast genome. The genome contains the coded instructions necessary for the organism to build and maintain itself. Genomics is the study of an organism’s genome, or genetic material. Genomics emerged as a new field of molecular biology where novel technologies were exploited in order to understand the complex, biological function of the genome. It revealed the static sequences of genes. The molecular biology revolution and the advent of genomic technologies are facilitating rapid advances in our understanding of the molecular details of cell and tissue function. These advances have the potential to transform toxicological and clinical practice, and are likely to lead to the supplementation or replacement of traditional biomarkers of cellular integrity, cell and tissue homeostasis, and morphological alterations that result from cell damage or death. The increasing amount of genomic and molecular information is the basis for understanding higher-order biological systems, such as the cell and the organism, and their interactions with the environment, as well as for medical, industrial and other practical applications (Kanehisa et al., 2006). The knowledge of full genomes has created the possibility for the field of functional genomics, mainly concerned with patterns of gene expression during various conditions. Microarray and bioinformatics are the most important tools of genomics. A major branch of genomics concerns with sequencing genomes of various organisms. In 1972, Walter Fiers and his team at the Laboratory of Molecular Biology of the University of Ghent (Ghent, Belgium) were the first to determine the sequence of a gene: the gene for bacteriophage MS2 coat protein. In 1976, the team determined the complete nucleotide-sequence of bacteriophage MS2-RNA. The first DNA-based genome to be sequenced in its entirety was that of bacteriophage -X174; (5,368 bp), sequenced by Frederick Sanger (Sanger et al., 1977). The first free-living organism to be sequenced was that of Haemophilus influenzae (1.8 Mb) in 1995, and since then genomes are being sequenced at a rapid pace.

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A rough draft of the human genome was completed by the Human Genome Project in early 2001, creating much fanfare. As of January 2005, the complete sequence was known of about 1,000 viruses, 220 bacterial species and roughly 20 eukaryote organisms, of which about half are fungi. Most of the bacteria whose genomes have been completely sequenced are problematic disease-causing agents, such as Haemophilus influenzae. Of the other sequenced species, most were chosen because they were well-studied model organisms or promised to become good models. Yeast (Saccharomyces cerevisiae) has long been an important model organism for the eukaryotic cell, while the fruit fly Drosophila melanogaster has been a very important tool (notably in early premolecular genetics). The worm Caenorhabditis elegans is an often used simple model for multicellular organisms. The zebrafish Brachydanio rerio is used for many developmental studies on the molecular level and the flower Arabidopsis thaliana is a model organism for flowering plants. The Japanese pufferfish (Takifugu rubripes) and the spotted green pufferfish (Tetraodon nigroviridis) are interesting because of their small and compact genomes, containing very little non-coding DNA compared to most species. The mammals dog (Canis familiaris), brown rat (Rattus norvegicus), mouse (Mus musculus; Lein et al., 2007) and chimpanzee (Pan troglodytes) are all important model animals in medical research. Genomics has the potential to revolutionize the practice of medicine, but despite significant scientific advances, very few genomics-based tests or treatments have reached consumers. The application of this genomics revealed that gene expression profiles induced by single drugs and the ones induced by the combination of the same drugs can be entirely different. The increasing amount of genomic and molecular information is the basis for understanding higher-order biological systems, such as the cell and the organism, and their interactions with the environment, as well as for medical, industrial and other practical applications. (Kanehisa et al., 2006). The combination of genomic technologies with a knowledge of gene sequence and sequence conservation has made available markers that facilitate the correlation of genetic variation with biological outcomes, and “-omic” technologies allow efficient biochemical characterization of functional pathways – providing new markers of the susceptibility of individuals to different diseases and response to specific therapies. The powerful -omic technologies allow efficient monitoring of gene transcripts, proteins, and intermediary metabolites, making it possible to monitor a large number of key cellular pathways simultaneously. This has enabled the identification of key biomarkers and signaling molecules associated with cell growth, cell death, and cellular metabolism. These new markers facilitate monitoring of functional disturbance, molecular and cellular damage, and damage-response (MacGregor, 2004). Over the past two decades, advances in genomic technology have allowed laboratories to generate vast amounts of biological data. These data include gene sequences, protein structures, information on gene expression and metabolic pathways. Automated instrumentation has enabled large volumes of data to be generated and automatically stored in computer databases, and this data has as many different formats as there are instruments. In addition to the new information gathered

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Transcriptome and Transcriptomics

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from genomic technologies, pharmaceutical and biotechnology companies have large amounts of “legacy data” – data inherited from their own and other sources on chemical structures and properties of compounds, and clinical, phenotypic and toxicological information. Most of this is stored in older types of databases designed for the particular type of data, and a major computational challenge is to integrate the new genomic information with current database systems in order to facilitate decision-making. Molecular biology and genetics has excelled in the creation of molecular diagnostics to identify and characterize disease processes. In the case of many diseases it has markedly altered clinical practice. In acute care settings it has led to increasingly accurate diagnosis and has impacted clinical treatment and outcomes. In HIV disease management, viral load and resistance genotyping has altered patient treatment decisions. Molecular diagnostics has potential role in multifactorial disease treatment such as breast cancer and this is an example of targeting a therapy to a specific genetic profile (Pegram and Slamon, 2000), however, Slamon and his co-workers demonstrated that amplification of the HER-2/neu gene was a significant predictor of both overall survival and time to relapse in patients with breast cancer. Similarly it took almost 20 years from the molecular understanding of the role of the bcr-abl fusion protein to the development of the therapeutic Gleevec. Nonetheless gene based testing technologies clearly offer a great advantage in identifying patient populations which can benefit from therapy. More importantly, the clear demonstration that one can proceed from a genetics-based disease understanding to therapeutic efficacy is a critical learning. While the impact early in the discovery phase is most significant, the application of genomics can contribute throughout the drug development process from initial discovery all the way through life cycle management.

2.4 Transcriptome and Transcriptomics Transcriptomics refers to the comprehensive scanning of the nearly fifty thousand currently known genes that are transcribed into RNA molecules from the three-billion-letter human genome. Each cell utilizes (expresses) different genes at different times in its development and under different physiological conditions. In general, tissues express similar sets of genes that can be used to identify those tissues in the absence of any other information. For example, the brain expresses about 30% of all of the known genes; those specific transcripts are different from the transcribed genome in the heart. We can therefore define molecular signatures based on expression profiles, and these profiles can then be used to automatically separate normal cells or tissues into their correct category. This method of expression profiling can also be done with disease states. In Alzheimer’s disease, neurons show altered gene expression patterns when compared to normal neurons. This information can be used as molecular diagnostic criteria in the absence of histopathology for tangles because it is, in fact, a surrogate marker of that cellular condition. Transcriptomics builds upon the more classical study of gene function where Northern blots are used for transcriptional analysis of

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individual genes. However, instead of examining one gene at a time this new technology analyses in one single go the entire transcriptome, which is the full set of all messenger RNA molecules present at any given time in a defined population of cells. This reveals an instant picture of all genes being actively transcribed, and since the technique is a least partly quantitative it also tells about the expression level of each gene. For example, by comparing such transcriptional analyses from plants exposed to different environmental conditions it is possible to quickly identify all genes involved in the adaptive process. All genes and most proteins can be regarded as instruments for making up the biochemical composition and there by the physiological identity of an organism. The same can be done for clinical states that may be less obvious or for which there does not exist a diagnostic marker (Example Autism). This approach will also facilitate testing in a pre-symptomatic scenario to encourage early interventions that may result in better outcomes. Transcriptomics approach facilitates sub-classification of disorders that on the surface appear to be similar. This strategy has been shown elegantly for a number of disorders, primarily in oncology, where one can define outcome and survival rates and drug response rates based on an expression profile. The development of microarray technologies and other high-throughput strategies are becoming increasingly important in biology because they permit to monitor expression levels of thousands of genes in only one experiment. Availability of computational tools to compare and analyse these expression profiles in a suitable way for biological interpretation is need of the day (Subramanian et al., 2005). Microarray technologies at the transcriptomic level can be high throughput and excellent for diagnostic purposes, but often their dynamic range, informational and experimental complexities limit their utility as diagnostic tools as compared with other assessments of mRNA abundance e.g. real time RT-PCR. One of the early applications of microarray was in mode of action (MOA) studies for isoniazid (INH) an antibiotic effective in the control of tuberculosis. Studies of M. tuberculosis exposed to INH indicated up regulation of a number of genes relevant to the MOA. In addition to possibly increasing the number of targets by identifying genes within pathways, the generation of “signature transcriptome analysis (TA) profiles” may be useful in predicting the MOA of new chemical entities. This approach has been shown to be quite useful in the search for tumor-specific markers and in distinguishing potential therapeutic targets in neoplastic cells. Gene expression profiles of tumors, for example, facilitate identification of subsets of genes that function as prognostic disease markers or biologic predictors of therapeutic response, thus permitting clinicians to distinguish indolent from aggressive tumors which require immediate therapeutic intervention. Although microarray studies can reveal the relative amounts of different mRNAs in the cell, levels of mRNA are not directly proportional to the expression level of the proteins they code for. The number of protein molecules synthesized using a given mRNA molecule as a template is highly dependent on transcription-initiation features of the mRNA sequence; in particular, the ability of the promoter region is a key determinant in the recruiting of ribosomes for protein translation. A study of 158,807 mouse transcripts revealed that 4,520 of these transcripts form

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antisense partners that are base pair complementary to the exons of genes (Katayama et al., 2005). These results raise the possibility that significant numbers of “antisense RNA-coding genes” might participate in the regulation of the levels of expression of protein-coding mRNAs.

2.5 Proteome and Proteomics With the avalanche of genomic information and improvements in analytical technology, proteomics is becoming increasingly important for the study of many different aspects. Since proteins serve as important components of major signaling and biochemical pathways, studies at protein levels are essential to reveal molecular mechanisms underlying disease processes. Proteomics is the application of evolving technologies to analyze gene products, proteins, on a large scale. This concerns protein expression profiles, protein modifications and protein networks in relation to cell function and biological processes e.g. development, health and disease (Macaulay et al., 2005). With the mapping of the human genome, proteomics has rapidly emerged as an exciting new field of research, one that complements rather than replaces genomics. The genome holds all of an organism’s genes, which carry the codes for the proteins that it needs to function. Proteomics is the integrated study of proteins and their biological functions and processes. It allows the study of protein structure and protein–protein interactions, and today, this branch of science is becoming a major tool in biomedical and drug development research. Since proteins play a central role in the life of an organism, proteomics is instrumental in discovery of biomarkers that indicate a particular disease. A surprising finding of the Human Genome Project is that there are far fewer protein-coding genes in the human genome than proteins in the human proteome (20,000–25,000 genes vs. about 1,000,000 proteins). The human body may contain more than 2 million proteins, each having different functions (http://en.wikipedia.org/wiki/Proteomics). The protein diversity is thought to be due to alternative splicing and post-translational modification of proteins. The discrepancy implies that protein diversity cannot be fully characterized by gene expression analysis, thus proteomics is useful for characterizing normal and abnormal cellular processes. To catalog all human proteins, their functions and interactions is a great challenge for scientists and an international collaboration with these goals is co-ordinated by the Human Proteome Organization (HUPO). Most proteins function in collaboration with other proteins, and one goal of proteomics is to identify which proteins interact. This often gives important clues about the functions of newly discovered proteins. Several methods are available to probe protein–protein interactions. The traditional method is yeast two-hybrid analysis. New methods include protein microarrays, immunoaffinity chromatography followed by mass spectrometry, and combinations of experimental methods such as phage display and computational methods

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(Wu et al., 2005). Proteomics uses various technologies such as (a) One- and twodimensional gel electrophoresis to identify the relative mass of a protein and its isoelectric point (b) X-ray crystallography (c) Nuclear magnetic resonance to characterize the three-dimensional structure of peptides and proteins (d) low-resolution techniques such as circular dichroism (e) Fourier transform infrared spectroscopy and small angle X-ray scattering (SAXS) to study the secondary structure of proteins and (f) Mass spectrometry (no-tandem), often MALDI-TOF is used to identify proteins by peptide mass finger printing. Protein profiling with MALDI-TOF MS can be of high use in clinical diagnostics. Affinity chromatography, yeast two hybrid techniques, fluorescence resonance energy transfer (FRET), and Surface Plasma Resonance (SPR) are used to identify protein–protein and protein-DNA binding reactions. X-ray Tomography is used to determine the location of labeled proteins or protein complexes in an intact cell. Soft ware based image analysis is utilized to automate the quantification and detection of spots within and among gels samples. While this technology is widely utilized, the intelligence has not been perfected yet. Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling (Nishizuka et al., 2003). The molecular biology revolution and the advent of genomic and proteomic technologies are facilitating rapid advances in our understanding of the molecular details of cell and tissue function. These advances have the potential to transform toxicological and clinical practice and are likely to lead to the supplementation or replacement of traditional biomarkers of cellular integrity, cell and tissue homeostasis, and morphological alterations that result from cell damage or death. Elucidation of cellular responses to molecular damage, including evolutionarily conserved inducible molecular defense systems, suggests the possibility of new biomarkers based on molecular responses to functional perturbations and cellular damage. Our improved understanding of the molecular basis of various pathologies suggests that monitoring specific molecular responses may provide improved prediction of human outcomes. Responses that can be monitored directly in the human should provide “bridging biomarkers” that may eliminate much of the current uncertainty in extrapolating from laboratory models to human outcome. More importantly the evaluation of one or a few biomarkers is severely limited by the fact that many disease syndromes share inflammatory components or involve the remodelling of extracellular matrix proteins common to many disease processes (e.g. fibrosis). Some of the diseases have characteristic biomarkers. These biomarkers are used by the physicians for diagnosis. Glycogen synthesis kinase has been a leader in advocating asystematic, highly parallel, combinatorial approach to assemble “disease specific signatures”, as well as resolving patterns in response to therapeutic agents. In Alzheimer’s disease, elevations in beta secretase creates amyloid/betaprotein, that causes plaque to build up in the patient’s brain, which causes dementia (Hye et al., 2006). Targeting this enzyme decreases the amyloid/beta-protein and so slows the progression of the disease. A procedure to test for the increase in amyloid/beta-protein is immunohistochemical staining, in which antibodies bind

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to specific antigens or biological tissue of amyloid/beta-protein. Heart disease is commonly assessed using several key protein based biomarkers (Vasan, 2006). Standard protein biomarkers for CVD include interleukin-6, interleukin-8, serum amyloid A protein, fibrinogen, and troponins. cTnI cardiac troponin I increases in concentration within 3–12 h of initial cardiac injury and can be found elevated days after an acute myocardial infarction. A number of commercial antibody based assays as well as other methods are used in hospitals as primary tests for acute myocardal infarction. Proteomic analysis of kidney cells and cancerous kidney cells is producing promising leads for biomarkers for renal cell carcinoma and developing assays to test for this disease (Perroud et al., 2006). In kidney-related diseases, urine is a potential source for such biomarkers. Identification of urinary polypeptides as biomarkers of kidney-related diseases allows to diagnose the severity of the disease several months before the appearance of the pathology (Rogers et al., 2003). Proteomic technologies rely on the ability to separate a complex mixture so that individual proteins are more easily processed with other techniques. Wellknown methods include low-throughput sequencing through Edman degradation. Higher-throughput proteomic techniques are based on mass spectrometry, commonly peptide mass fingerprinting on simpler instruments, or De novo repeat detection sequencing on instruments capable of more than one round of mass spectrometry. Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact on biology and medicine (Aebersold and Mann, 2003). Identifying all protein–protein interactions in an organism is a major objective of proteomics. A related goal is to know which protein pairs are present in the same protein complex. High-throughput methods such as yeast two-hybrid (Y2H) and affinity purification coupled with mass spectrometry (APMS) have been used to detect interacting proteins on a genomic scale. However, both Y2H and APMS methods have substantial false-positive rates. Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction. Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship. Many proteins participate in cellular processes as members of protein complexes of varying size. It is believed that combinatorial interactions among proteins serve as an important basis for the biological complexity of higher organisms. Therefore, increased knowledge about protein–protein interactions and protein complexes will greatly aid our understanding of protein function (Blagoev et al., 2003; Zhang et al., 2004). Advances in mass spectrometry (MS), such as surface-enhanced laser desorption ionization (SELDI), hold great promise for early ovarian cancer detection through proteomic profiling of patient serum. Because thousands of proteins and peptides can be characterized and quantified at the same time, large amount of valuable data are obtained for identifying characteristic and effective biomarkers for ovarian cancer detection. Several advanced data mining algorithms have been reported

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to be promising for diagnosis of early-stage ovarian cancer (Petricoin et al., 2002; Wulfkuhle et al., 2003; Tirumalai et al., 2003; Zhu et al., 2003; Yanagisawa et al., 2003; Pan et al., 2005). However, considerable controversy has been generated, and there remain some critical issues such as reproducibility and robustness of these methods, which make the proteomic profiling approach less congenial for achieving definite diagnosis (Diamandis, 2004; Baggerly et al., 2005; Ransohoff, 2005). Kristiansen et al. (2004) have carried out a comprehensive characterization of human bile to define the bile proteome by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. The strategy can be applicable for a detailed proteomic analysis of most body fluids. In combination with “tagging” approaches for differential proteomics, the proteomics approach could serve to identify cancer biomarkers from any body fluid (Baggerly et al., 2005). In terms of the drug development process emphasis has been placed upon staging and progression rather than diagnosis. Recent development of highly multiplexed plate, microbead and protein microarray technologies (Liotta et al., 2005) with larger numbers of analytes (with clinical relevance) gave rise to applications such as protein profiling of plasma from complex disease states. Protein microarray and assay detection systems based on this technology were assumed to emerge as the correlates of DNA microarrays in the identification, quantitation and functional analysis of proteins. In concept, protein microarray is a fairly simple process, antibodies or other affinity reagents are attached to a surface and a cell lysate or serum, is added. After a period of time to allow the antigens present to bind to their cognate antibodies, bound antigen is detected using fluorescently tagged secondary antibodies. Alternatively lysates containing fluorescently tagged or radioactively labelled proteins can be added directly to the array to permit direct detection of the analyte. Currently, “cytokine-chemokine array” strategies appear to be more robust, since a number of well characterized reagents and commercial arrays are available and many disease syndromes display characteristic patterns of cytokine and chemokine expression. Other functional protein arrays are being used to study protein–protein, protein-nucleic acid, protein-allergen, protein-carbohydrate, protein-lipid, enzymesubstrate, and protein-drug interactions. While antibody reagents are not available for most proteins, a number of initiatives have been undertaken to expand the number of antibodies, aptamers and defined proteins for use in quantitative and function based assays. One of the most promising developments to come from the study of human genes and proteins has been the identification of potential new drugs for the treatment of disease. This relies on genome and proteome information to identify proteins associated with a disease. When a particular protein is implicated in a disease, its 3D structure provides the information to design drugs to interfere with the action of the protein. A molecule that fits the active site of an enzyme, but cannot be released by the enzyme, will inactivate the enzyme. This is the basis of new drug-discovery tools, which aim to find new drugs to inactivate proteins involved in disease. The genetic differences among individuals can be exploited to develop personalized drugs that are more effective for the individual (Arora et al., 2005).

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A computer technique which attempts to fit millions of small molecules to the three-dimensional structure of a protein is called “virtual ligand screening”. The computer rates the quality of the fit to various sites in the protein, with the goal of either enhancing or disabling the function of the protein, depending on its function in the cell. A good example of this is the identification of new drugs to target and inactivate the HIV-1 protease. The HIV-1 protease is an enzyme that cleaves a very large HIV protein into smaller, functional proteins. The virus cannot survive without this enzyme; therefore, it is one of the most effective protein targets for killing HIV. An important use of proteomics is using specific protein biomarkers to diagnose disease. Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions will be critical for developing the most effective diagnostic techniques and disease treatments in the future .The long-term challenge of proteomics is enormous: to define the identities, quantities, structures and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts. One critical step in tackling this goal is the generation of sets of clones that express a representative of each protein of a proteome in a useful format, followed by the analysis of these sets on a genome-wide basis. Such studies enable genetic, biochemical and cell biological technologies to be applied on a systematic level, leading to the assignment of biochemical activities, the construction of protein arrays, the identification of interactions, and the localization of proteins within cellular compartments (Phizicky et al., 2003).

2.6 Metabolome and Metabolomics Since the emergence of genomics, several other “omic” techniques have come to the front and promising one is “metabolomics” (Schmidt, 2004). Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signalling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism (Oliver et al., 1998). Metabolomics (also referred to as metabonomics) is the comprehensive study of the metabolome, the repertoire of biochemicals (or small molecules) present in cells, tissue, and body fluids. One of the major challenges of this approach, as with other “omic” technologies, is that the metabolome is context-dependent and will vary with pathology, developmental stage and environmental factors. Following the rapid developments in genome, transcriptome and proteome technologies, there is a growing interest in metabolome research. The study of metabolism at the global or “-omics” level is a new but rapidly growing field that has the potential to have an impact upon medical practice as the metabolic state reflects what has been encoded by the genome and modified by environmental factors (Kaddurah-Daouk, 2006). In January 2007, scientists at the University of Alberta and the University of Calgary completed the first draft of the human metabolome (Wishart et al., 2007).

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They have catalogued and characterized 2,500 metabolites, 1,200 drugs and 3,500 food components that can be found in the human body (Harrigan and Goodacre, 2003). In animals and humans, metabolic profiling of blood and urine components to characterize toxicity and disease states such as inborn errors of metabolism has been ongoing since the introduction of gas-chromatography mass-spectrometry (GC-MS) in the mid-1960s. Techniques such as NMR spectroscopy can be used for a very wide range of components of blood or urine, and advanced data analysis techniques locate the key descriptors (biomarkers) of effect. In addition to NMR, other spectroscopic methods such as MS, GC/MS can produce metabolic profiles or signatures of toxicity, disease or drug efficacy. Thus, the possibility of globally profiling the metabolome of an organism is a genuine analytical challenge, as by definition this must also take into consideration all relevant factors that influence metabolism. Despite these challenges, the approach has already been applied to understand the metabolism in a range of animal models, and has more recently started to be projected into the clinical situation (Brindle et al., 2002). The use of metabolomic data to predict the health trajectories of individuals will require bioinformatic tools and quantitative reference databases. These databases containing metabolite profiles from the population must be built, stored and indexed according to metabolic and health status. Building and annotating these databases with the knowledge to predict how a specific metabolic pattern from an individual can be adjusted with diet, drugs and lifestyle to improve health represents a logical application of the biochemistry knowledge that the life sciences have produced over the past 100 years (German et al., 2005). Using information from the completely sequenced genomes and known metabolic pathways of various microorganisms, there are ongoing attempts to completely model the metabolism of a cell or microorganism. In metabolomics research, there are several steps between the sampling of the biological condition under study and the biological interpretation of the results of the data analysis (German et al., 2002). First, the biological samples are extracted and prepared for analysis. Subsequently, different data pre-processing steps (Fiehn, 2002) are applied in order to generate “clean” data in the form of normalized peak areas that reflect the (intracellular) metabolite concentrations. These clean data can be used as the input for data analysis. However, it is important to use an appropriate data pretreatment method before starting data analysis. Data pretreatment methods convert the clean data to a different scale (for instance, relative or logarithmic scale). Hereby, they aim to focus on the relevant (biological) information and to reduce the influence of disturbing factors such as measurement noise. Procedures that can be used for data pretreatment are scaling, centering and transformations. The ability to use metabolomics approaches for classification and mechanistic studies may influence and augment our ability to study and address the aging process scientifically and clinically (Kristal and Shurubor, 2005). Metabolic profiling (especially of urine or blood plasma samples) can be used to detect the physiological changes caused by toxic insult of a chemical (or mixture of chemicals). In many cases, the observed changes can be related to specific syndromes, e.g. a specific lesion in liver or kidney. This is of particular relevance

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to pharmaceutical companies wanting to test the toxicity of potential drug candidates: if a compound can be eliminated before it reaches clinical trials on the grounds of adverse toxicity, it saves the enormous expense of the trials (Lindon et al., 2004). Metabolites are the key regulators of systems homeostasis. As such, concentration changes of specific groups of metabolites may reflect systemic responses to environmental, therapeutic or genetic interventions. Thus, the study of metabolites is a powerful tool for the characterization of complex phenotypes as well as for the development of biomarkers for specific physiological responses. Metabolic signatures provide prognostic, diagnostic, and surrogate markers for a disease state; the ability to subclassify disease; biomarkers for drug response phenotypes (pharmacometabolomics); and information about mechanisms of disease. Indeed, sophisticated metabolomic analytical platforms and informatics tools have recently been developed that make it possible to define initial metabolic signatures for several diseases (Kaddurah-Daouk, 2006). Therefore, metabolomics is a valuable platform for studies of complex diseases and the development of new therapies, both in non-clinical disease model characterization and clinical settings. Applications to preclinical drug safety studies are illustrated by the Consortium for Metabolomic Toxicology, a collaboration involving several major pharmaceutical companies (Brindle et al., 2002). The use of metabolomics in disease diagnosis and therapy monitoring and the concept of pharmaco-metabonomics as a way of predicting an individual’s response to treatment can be studied (Ellis and Goodacre, 2006). Combination drug therapies with individualized optimization are likely to become a major focus. Metabolomics incorporates the most advanced approaches to molecular phenotype system readout and provides the ideal theranostic technology platform for the discovery of biomarker patterns associated with healthy and disease states, for use in personalized health monitoring programs and for the design of individualized interventions (German et al., 2003). Today, clinicians capture only a very small part of the information contained in the metabolome, as revealed by a defined set of blood chemistry analyses to define health and disease states. Examples include measuring glucose to monitor diabetes and measuring cholesterol for cardiovascular health. Such a narrow chemical analysis could potentially be replaced in the future with a metabolic signature that captures global biochemical changes in disease and upon treatment.

2.7 Integration of Omics Developing a new drug is a tedious and expensive undertaking. The recently developed high-throughput experimental technologies, encompassing genomics, transcriptomics, proteomics and metabolomics provide for the first time ever the means to comprehensively monitor the disease processes at the molecular level. They increase substantially the number of proteins/genes that can be detected simultaneously and have the potential to relate complex mixtures to complex effects in the form of gene/protein expression profiles. The primary aim of “omic” technologies is the nontargeted identification of all gene products (transcripts, proteins, and

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metabolites) present in a specific biological sample. By their nature, these technologies reveal unexpected properties of biological systems. A second and more challenging aspect of omic technologies is the refined analysis of quantitative dynamics in biological systems. The “-omics” technologies facilitate the systematic characterization of a drug target’s physiology, thereby helping to reduce the typically high attrition rates in discovery projects, and improving the overall efficiency of pharmaceutical research processes. Currently, the bottlenecks for taking full advantage of these experimental technologies are the rapidly growing volumes of automatically produced biological data and lack of scalable database systems and computational tools (Fischer, 2005). High-throughput analysis is essential considering data at the “omic” level. There are four major types of high-throughput measurements that are commonly performed: genomic SNP analysis (i.e., the large-scale genotyping of single nucleotide polymorphisms), transcriptomic measurements (i.e., the measurement of all gene expression values in a cell or tissue type simultaneously), proteomic measurements (i.e., the identification of all proteins present in a cell or tissue type), and metabolomic measurements (i.e., the identification and quantification of all metabolites present in a cell or tissue type). Each of these four is distinct and offers a different perspective on the processes underlying disease initiation and progression as well as on ways of predicting, preventing, or treating disease (Venkatesh and Harry, 2002). Genomic SNP genotyping measures a person’s genotypes for several hundred thousand single nucleotide polymorphisms spread throughout the genome. These SNPs tend to be quite common (with typically at least 5% of the population having at least one copy of the less frequent allele), and not strictly causal of the disease. Rather, SNPs can act in unison with other SNPs and with environmental variables to increase or decrease a person’s risk of a disease. This makes identifying important SNPs difficult; the variation in a trait that can be accounted for by a single SNP is fairly small relative to the total variation in the trait. Even so, because genotypes remain constant (barring mutations to individual cells) throughout life, SNPs are potentially among the most useful measurements for predicting risk. Transcriptomic measurements (often referred to as gene expression microarrays or “gene chips” are the oldest and most established of the high-throughput methodologies). Gene expression levels influence traits more directly than SNPs, and so significant associations are easier to detect. While transcriptomic measures are not as useful for pre-disease prediction (because a person’s gene expression levels very far in advance of disease initiation are not likely to be informative because they have the potential to change so significantly), they are very well-suited for either early identification of a disease (i.e., finding people who have gene expression levels characteristic of a disease but who have not yet manifested other symptoms) or classifying patients with a disease into subgroups (by identifying gene expression levels that are associated with either better or worse outcomes or with higher or lower values of some disease phenotype). Proteomics is similar in character to transcriptomics. Like transcriptomic measures, though, proteomic measures are excellent for early identification of disease or classifying people into subgroups. As with proteomics, the metabolites are measured

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in a very fast serial process. NMR is typically used to both identify and quantify metabolites. This technology is newer and less frequently used than the other technologies, but similar caveats apply. Measurements of metabolites are dynamic as are gene expression levels and proteins, and so are best suited for either early disease detection or disease subclass identification. The new omics technologies seem set to fulfill their huge expectations and in combination they might prove extremely valuable in functional gene analyses. Genomic and transcriptomic studies are mostly conducted by DNA microarray technologies. Proteomics and metabolomics have no standardized procedures yet, but usually, proteome analysis is done by two-dimensional gel electrophoresis, Liquid chromatography-mass spectrometry, while metabolome analysis is conducted through Gas chromatography-mass spectrometry, Liquid chromatographymass spectrometry and Liquid chromatography – Nuclear magnetic resonance and MALDI-TOF. Transcriptomics provides the tool for deciphering gene expression networks, and proteomics links these networks to protein products. The third crucial partner is metabolomics, which defines the metabolic network(s) linked to gene expression. NMR and mass spectrometry enable the broad screen analysis of the metabolome and its transformation pathways, transcending classical targeted metabolic studies. Omics will not only have an impact on our understanding of biological processes, but the prospect of more accurately diagnosing and treating disease will soon become a reality (Loughlin, 2007). Development of microarrays has permitted global measurement of gene expression at the transcript level and provided a glimpse into the coordinated control and interactions between genes (Mutch et al., 2002). Additional “transcriptomic” and other “omic” analyses will further advance medical research. Protein profiling (proteome), more comprehensive analysis of gene function by knocking down or out of candidate genes (phenome), protein–protein interaction (interactome), and protein cellular and subcellular localization (localizome) are all needed for a deeper understanding of the development, physiology, and pathology of any organ or tissue. Each technique will collectively contribute to the diagnosis, prevention, and therapy of the great number of heart diseases (Coen et al., 2004). The unprecedented advances in molecular biology during the last two decades have resulted in a dramatic increase in knowledge about gene structure and function, an immense database of genetic sequence information, and an impressive set of efficient new technologies for monitoring genetic sequences, genetic variation, and global functional gene expression. These advances have led to a new sub-discipline of toxicology: “toxicogenomics”. We define toxicogenomics as“the study of the relationship between the structure and activity of the genome (the cellular complement of genes) and the adverse biological effects of exogenous agents”. Toxicogenomics help to monitor the expression of multiple genes, proteins, and metabolites simultaneously. It combines new technologies in genomics, proteomics, and metabolomics (Fig. 2.2) with traditional tools of pathology and toxicology to study biological response to drugs and other environmental xenobiotics. The biological response to environmental exposure is so complex and involves so many interactive factors that the use of a systems biology analytical approach is required.

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Fig. 2.2 Integrating omics technology: One of the challenges of systems biology is to integrate genomics, proteomic, transcriptomic, and metabolomic information to give a more complete picture of living organisms

These new technologies will lead to new families of biomarkers that permit characterization and efficient monitoring of cellular perturbations, provide an increased understanding of the influence of genetic variation on toxicological outcomes, and allow definition of environmental causes of genetic alterations and their relationship to human disease. The broad application of these new approaches will likely erase the current distinctions among the fields of toxicology, pathology, genetic toxicology, and molecular genetics. Instead, a new integrated approach will likely emerge that involves a comprehensive understanding of genetic control of cellular functions, and of cellular responses to alterations in normal molecular structure and function (Aardema and MacGregor, 2002). It was realized however, that many, possibly thousands of other gene polymorphisms might result in minor deviations in nutritional biochemistry, where only marginal or additive effects would result from these deviations. The tools to study the physiological impact were not available at the time and are only now becoming available. “Nutrigenomics” is the application of the sciences of genomics, transcriptomics, proteomics and metabolomics to human nutrition, especially the relationship between nutrition and health. Nutrigenomics is associated with the issue of personalized nutrition, since claims are being made that differences in genotype should result in differences in the diet and health relationship. The recent advances in nutrigenomics studies are owed to the completion of human genome project and the new biomics technologies that provide means for the simultaneous determination of the expression of many thousands of genes at the mRNA (transcriptomics), metabolites (metabolomics) and protein (proteomics) levels. The role of metabolomics in nutrigenomics (German et al., 2003) requires understanding, and ultimately regulating, a multitude of nutrient-related interactions at the gene, protein and metabolic levels. These new disciplines and their attendant technologies are changing the paradigms of health research. A number of genetic variations have been shown to increase the susceptibility to diet-related diseases. These include variants that have been associated with Type 2 diabetes mellitus, obesity, cardiovascular diseases, some autoimmune diseases and cancers. Nutrigenetics aims to study these susceptible genes and provide dietary interventions for individuals at risk of such diseases. Hyperlipidemia is usually associated with atherosclerosis and coronary heart disease (Kaput, 2004). Therapy includes lifestyle changes as alterations in the patient’s

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diet, physical activity and treatment with pharmaceuticals as statins. However, individuals respond differently to the treatment. This was attributed to genetic variations within the population. Genetic variations in genes encoding for apolipoproteins, some enzymes and hormones can alter individual sensitivity to developing cardiovascular diseases (Ordovas and Mooser, 2004). Nutrients can contribute to the development of cancers especially colon, gastric and breast cancer. Several gene variants have been identified as susceptibility genes. One example is the N-Acetyltransferase (NAT) gene. Studies have shown that the NAT2 fast acetylator genotype had a higher risk of developing colon cancer in people who consumed relatively large quantities of red meat (Mutch et al., 2005). The advent to access comprehensive sets of information (i.e., genome, transcriptome, proteome, interactome, phenome, and localizome) has brought a new way of global thinking to biological questions, and analysis using these sets is increasingly the choice for many investigations. Although these approaches may appear as a substitute for the more traditional “reductionist” approach that tackles one or few genes or gene products at one time, they are, in reality, complementary and have the potential to greatly enhance the traditional approach (Abu-Issa and Kirby, 2004). Less than a decade ago, describing the complexity of chemical behavior in biological systems was severely limited because realistic models presented combinatorial and other problems beyond the capabilities of most computers. In the field of bioinformatics, for example, major advances were made not from faster statistical analysis of data after the acquisition, but from the integration of computational and data acquisition technologies. It is now possible to consider how to evaluate the vast amounts of information generated by “omic” technologies using data-mining tools made possible by rapid advances in computational storage capacity and speed. Within the systems biology framework, functional analyses at the level of gene expression (transcriptomics), protein translation (proteomics), and, more recently, the metabolite network (metabolomics) have become increasingly popular. Metabolomics experiments aim to quantify all metabolites in a cellular system (cell or tissue) under defined states and at different time points so that the dynamics of any biotic, abiotic, or genetic perturbation can be accurately assessed. With the progress in genetics, biochemical disorders with high nutritional relevance were linked to a genetic origin. The next generation omics will include genome resequencing, methylation analysis, mRNA tag profiling, small RNA identification, transcriptome sequencing and also the functional elements (Fig. 2.3). The “omic,” or comprehensive, approach can yield mountains of new information in a relatively short time. Progress in applying the “omic” approach is still in its early exponential phase and does not look like it will plateau any time soon.

2.8 Conclusion The technology platform of genomics, proteomics and metabolomics (“-omic-” technologies) are high-throughput technologies. They increase substantially the number of proteins/genes that can be detected simultaneously and have the potential

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Fig. 2.3 The next generation omics will include all the outcomes after integrating omics

to relate complex mixtures to complex effects in the form of gene/protein expression profiles. By their nature, these technologies reveal unexpected properties of biological systems. A second and more challenging aspect of omic technologies is the refined analysis of quantitative dynamics in biological systems. Several recent technological advances now make it possible to develop molecular profiles using genomic, proteomic, and metabolomic methods in order to identify the effects that chemicals may have on living organisms or the environment. Although the technology continues to change and improve, conducting these types of analyses is no longer a question of capability. In summary, the new omics technologies seem set to fulfill huge expectations and in combination they might prove extremely valuable in functional gene analyses. It is, however, necessary to further standardize and automate the methods of especially proteomics and metabolomics in order to make efficient and reproducible high-throughput analyses. Omics will not only have an impact on our understanding of biological processes, but the prospect of more accurately diagnosing and treating disease will soon become a reality. Advances in “omics” technologies have the potential to revolutionize our approach to disease diagnosis, prognostication and development of novel therapeutics. However, the promise of rapid advances in medicine “from the lab bench to the bedside” has not manifested as of yet. Indeed it appears that the translational applications of genomic-based research have preceded the development of both (i) a conceptual framework for disease understanding and (ii) effective tools that can exploit the vast amounts of data derived from these efforts. In reality great progress has been made, however understanding processes such as disease progression (or

References

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drug response) requires systematic insight into dynamic (and temporal) differences in gene regulation, interaction and function (Bilello, 2005). Omics is a progressive and useful concept in biology. It can revolutionize the way biology is done and how we see life in the future. However, omics fields itself is undergoing change. Some omics do not have livable niche. As time goes by, practically useful omics will survive and go into the mainstream biology while some others will die out until revived in an unexpected way. The omic methods will be powerful aids in understanding biology and advancing clinical practice, but it was never going to be as easy as some led us to believe.

References Aardema, M.J., MacGregor, J.T., 2002, Toxicology and genetic toxicology in the new era of “toxicogenomics”: impact of “-omics” technologies. Mutat Res 499:13–25. Abu-Issa, R., Kirby, M.L., 2004, Take heart in the age of “Omics”. Circ Res 95:335. Aebersold, R., Mann, M., 2003, Mass spectrometry-based proteomics. Nature 422:198–207. Arora, P.S., Yamagiwa, H., Srivastava, A., Bolander, M.E., Sarkar, G., 2005, Comparative evaluation of two two-dimensional gel electrophoresis image analysis software applications using synovial fluids from patients with joint disease. J Orthop Sci 10:160–166. Baggerly, K.A., Morris, J.S., Edmonson, S.R., Coombes, K.R., 2005, Signal in noise: evaluating reported reproducibility of serum proteomic tests for ovarian cancer. J Natl Cancer Inst 97: 307–309. Bilello, J.A., 2005, The agony and ecstasy of “OMIC” technologies in drug development. Curr Mol 5:39–52. Blagoev, B., Kratchmarova, I., Ong, S.E., Nielsen, M., Foster, L.J., Mann, M., 2003, A proteomics strategy to elucidate functional protein–protein interactions applied to EGF signaling. Nat Biotechnol 21:315–318. Brindle, J.T., Antti, H., Holmes, E., Tranter, G., Nicholson, J.K., Bethell, H.W.L., Clarke, S., Schofield, P.M., McKilligin, E., Mosedale, D.E., Grainger, D.J., 2002, Rapid and non-invasive diagnosis of the presence and severity of coronary heart disease using 1H NMR-based metabonomics. Nature Med 8:1439–1444. Coen, M., Ruepp, S.U., Lindon, J.C., Nicholson, J.K., Pognan, F., Lenz, E.M., Wilson, I.D., 2004, Integrated application of transcriptomics and metabonomics yields new insight into the toxicity due to paracetamol in the mouse. J Pharm Biomed Anal 35:93–105. Diamandis, E.P., 2004, Analysis of serum proteomic patterns for early cancer diagnosis: drawing attention to potential problems. J Natl Cancer Inst 96:353–356. Ellis, D.I., Goodacre, R., 2006, Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy. Analyst 131:875–885. Fiehn, O., 2002, Metabolomics the link between genotypes and phenotypes. Plant Mol Biol 48:155–171. Fiers, W., Contreras, R., Duerinck, F., Haegeman, G., Iserentant, D., Merregaert, J., Min Jou, W., Molemans, F., Raeymaekers, A., Van den Berghe, A., Volckaert, G., Ysebaert, M., 1976, Complete nucleotide-sequence of bacteriophage MS2-RNA – primary and secondary structure of replicase gene. Nature 260:500–507. Fischer, H.P., 2005, Towards quantitative biology: integration of biological information to elucidate disease pathways and to guide drug discovery. Biotechnol Annu Rev 11:1–68. German, J.B., Hammock, B.D., Watkins, S.M., 2005, Metabolomics: building on a century of biochemistry to guide human health. Metabolomics 1:3–9. German, J.B., Roberts, M.A., Fay, L., Watkins, S.M., 2002, Metabolomics and individual metabolic assessment: the next great challenge for nutrition. J Nutr 132:2486–2487.

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Omics Technology

German, J.B., Roberts, M.A., Watkins, S.M., 2003, Genomics and metabolomics as markers for the interaction of diet and health: lessons from lipids. J Nutr 133:2078S–2083S. Harrigan, G.G., Goodacre, R., 2003, Metabolic Profiling: Its Role in Biomarker Discovery and Gene Function Analysis. Kluwer Academic Publishers, Boston, ISBN 1-4020-7370-4. Hye, A., Lynham, S., Thambisetty, M., Causevic, M., Campbell, J., Byers, H.L., Hooper, C., Rijsdijk, F., Tabrizi, S.J., Banner, S., Shaw, C.E., Foy, C., Poppe, M., Archer, N., Hamilton, G., Powell, J., Brown, R.G., Sham, P., Ward, M., Lovestone, S., 2006, Proteome-based plasma biomarkers for Alzheimer’s disease. Brain 129:3042–3050. Kaddurah-Daouk, R., 2006, Metabolic profiling of patients with schizophrenia. PLoS Med 3:363. Kanehisa, M., Goto, S., Hattori, M., Aoki-Kinoshita, K.F., Itoh, M., Kawashima, S., Katayama, T., Araki, M., Hirakawa, M., 2006, From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357. Kaput, J., 2004, Diet-disease gene interactions. Nutrition 20:26–31. Katayama, S., Tomaru, Y., Kasukawa, T., Waki, K., Nakanishi, M., Nakamura, M., Nishida, H., Yap, C.C., Suzuki, M., Kawai, J., et al., 2005, “Antisense transcription in the mammalian transcriptome” by the RIKEN Genome Exploration Research Group and Genome Science Group (Genome Network Project Core Group) and the FANTOM Consortium. Science 309:1564–1566. Kristal, B.S., Shurubor, Y.I., 2005, Metabolomics: opening another window into aging. Sci Aging Knowl Environ, (26), pe 1. Kristiansen, T.Z., Bunkenborg, J., Gronborg, M., Molina, H., Thuluvath, P.J., Argani, P., Goggins, M.G., Maitra, A., Pandey, A., 2004, A proteomic analysis of human bile. Mol Cell Proteomics 3:715–728. Lein, E.S., Hawrylycz, M.J., Ao, N., Ayres, M., Bensinger, A., Bernard, A., Boe, A.F., Boguski, M.S., Brockway, K.S., Byrnes, E.J., et al., 2007, Genome-wide atlas of gene expression in the adult mouse brain. Nature 445:168–176. Lindon, J.C., Holmes, E., Nicholson, J.K., 2004, Metabonomics technologies and their applications in physiological monitoring, drug safety assessment and disease diagnosis. Biomarkers 9(1): 1–31. Liotta, L.A., Lowenthal, M., Mehta, A., Conrads, T.P., Veenstra, T.D., Fishman, D.A., Petricoin, E.F., III, 2005, Importance of communication between producers and consumers of publicly available experimental data. J Natl Cancer Inst 97:315–319. Loughlin, M.F., 2007, Using ‘omic’ technology to target Helicobacter pylori. Drug Discov 2: 1041–1051. Macaulay, I.C., Carr, P., Gusnanto, A., Ouwehand, W.H., Fitzgerald, D., Watkins, N.A., 2005, Platelet genomics and proteomics in human health and disease. J Clin Invest 115: 3370–3377. MacGregor, J.T., 2004, Biomarkers of cancer risk and therapeutic benefit: new technologies, new opportunities, and some challenges. Toxicol Pathol 1:99–105. Min Jou, W., Haegeman, G., Ysebaert, M., Fiers, W., (1972) Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein. Nature 237:82–88. Mutch, D.M., Berger, A., Mansourian, R., Rytz, A., Roberts, M.A., 2002, The limit fold change model: a practical approach for selecting differentially expressed genes from microarray data. BMC Bioinformatics 3:17. Mutch, D.M., Wahli, W., Williamson, G., 2005, Nutrigenomics and nutrigenetics: the emerging faces of nutrition. FASEB J 19:1602–1616. Nishizuka, S., Charboneau, L., Young, L., Major, S., Reinhold, W.C., Waltham, M., Kouros-Mehr, H., Bussey, K.J., Lee, J.K., Espina, V., Munson, P.J., Petricoin, E., 3rd, Liotta, L.A., Weinstein, J.N., 2003, Proteomic profiling of the NCI-60 cancer cell lines using new high-density reversephase lysate microarrays. Proc Natl Acad Sci USA 100:14229–14234. Oliver, S.G., Winson, M.K., Kell, D.B., Baganz, F., 1998, Systematic functional analysis of the yeast genome. Trends Biotechnol 16:373–378. Ordovas, J.M., Mooser, V., 2004, Nutrigenomics and nutrigenetics. Curr Opin Lipidol 15:101–108.

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Pan, S., Zhang, H., Rush, J., Eng, J., Zhang, N., Patterson, D., Comb, M.J., Aebersold, R., 2005, High throughput proteome screening for biomarker detection. Mol Cell Proteom 4: 182–190. Pegram, M., Slamon, D., 2000, Biological rationale for HER2/neu (c-erbB2) as a target for monoclonal antibody therapy. Semin Oncol 27:13–19. Perroud, B., Lee, J., Valkova, N., Dhirapong, A., Lin, P.Y., Fiehn, O., Kültz, D., Weiss, R.H., 2006, Pathway analysis of kidney cancer using proteomics and metabolic profiling. Mol Cancer 5:64–82. Petricoin, E.F., Ardekani, A.M., Hitt, B.A., Levine, P.J., Fusaro, V.A., Steinberg, S.M., Mills, G.B., Simone, C., Fishman, D.A., Kohn, E.C., Liotta, L.A., 2002, Use of proteomic patterns in serum to identify ovarian cancer. Lancet 359:572–577. Phizicky, E., Bastiaens, P.I., Zhu, H., Snyder, M., Fields, S., 2003, Protein analysis on a proteomic scale. Nature 422:208–215. Ransohoff, D.F., 2005, Lessons from controversy: ovarian cancer screening and serum proteomics. J Natl Cancer Inst 97:315–319. Rogers, M.A., Clarke, P., Noble, J., Munro, N.P., Paul, A., Selby, P.J., Banks, R.E., 2003, Proteomic profiling of urinary proteins in renal cancer by surface enhanced laser desorption ionization, and neural-network analysis: identification of key issues affecting clinical potential utility. Cancer Res 63:6971–6983. Sanger, F., Air, G.M., Barrell, B.G., Brown, N.L., Coulson, A.R., Fiddes, C.A., Hutchison, C.A., Slocombe, P.M., Smith, M., 1977, Nucleotide sequence of bacteriophage phi X174 DNA. Nature 265:687–695. Schmidt, C., 2004, Metabolomics takes its place as latest up-and-coming “omic”science. J Natl Cancer Inst 96:732–734. Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S., Mesirov, J.P., 2005, Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 102:15545–15550. Tirumalai, R.S., Chan, K.C., Prieto, D.A., Issaq, H.J., Conrads, T.P., Veenstra, T.D., 2003, Charaterization of the low molecular weight human serum proteome. Mol Cell Proteom 10:1096–1103. Vasan, R.S., 2006, Biomarkers of cardiovascular disease: molecular basis and practical considerations. Circulation 113:2335–2362. Venkatesh, T.V., Harry, B., 2002, Harlow integromics: challenges in data integration. Genome Biol 3:reports 4027.1–reports 4027.3. Wishart, D.S., Tzur, D., Knox, C., Eisner, R., Guo, A.C., Young, N., Cheng, D., Jewell, K., Arndt, D., Sawhney, S., et al., 2007, HMDB: the human metabolome database. Nucleic Acids Res 35:521–526. Wu, S.L., Kim, J., Hancock, W.S., Karger, B., 2005, Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR). J Proteome Res 4:1155–1170. Wulfkuhle, J.D., Liotta, L.A., Petricoin, E.F., 2003, Proteomic applications for the early detection of cancer. Nat Rev Cancer 3(4):267–275. Yanagisawa, K., Shyr, Y., Xu, B.J., Massion, P.P., Larsen, P.H., White, B.C., Roberts, J.R., Edgerton, M., Gonzalez, A., Nadaf, S., Moore, J.H., Caprioli, R.M., Carbone, D.P., 2003, Proteomic patterns of tumour subsets in non-small-cell lung cancer. Lancet 362:433–439. Zhang, Z., Bast, R.C., Jr., Yu, Y., Li, J., Sokoll, L.J., Rai, A.J., Rosenzweig, J.M., Cameron, B., Wang, Y.Y., Meng, XY., et al., 2004a, Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer. Cancer Res 64:5882–5890. Zhu, W., Wang, X., Ma, Y., Rao, M., Glimm, J., Kovach, J.S., 2003, Detection of cancer-specific markers amid massive mass spectral data. Proc Nat Acad Sci USA 100:14666–14671.

Chapter 3

Recombinant DNA Pharmaceuticals

Abstract Modern molecular medicine encompasses the utilization of many molecular biological techniques in the analysis of disease, disease genes and disease gene function. The study of disease genes and their function in an unaffected individual has been possible by the development of recombinant DNA and cloning techniques. The term gene cloning covers a range of techniques that makes it possible to manipulate DNA in a test tube and also to return it to living organism where it function normally. The tools of gene cloning includes vectors, genes and the enzymes. Overcoming the biological and methodological obstacles posed by cell factories to the production or rDNA pharmaceuticals is a main challenge in the further development of protein-based molecular medicine. Recombinant DNA technologies might have exhausted conventional cell factories and new production systems need to be deeply explored and incorporated into the production pipeline. On the other hand, a more profound comprehension of host cell physiology and stress responses to protein production would necessary offer improved tools (either at genetic, metabolic or system levels) to favour high yield and high quality protein production. Apart from the expected incorporation of unusual mammalian hosts such as transgenic animals or plants, microbial cells appear as extremely robust and convenient hosts, and gaining knowledge about the biological aspects of protein production would hopefully enhance the performance of such hosts beyond the current apparent limitations. In this regard, not only commonly used bacteria and yeasts but unconventional strains or species are observed as promising cell factories for forthcoming recombinant drugs. Their incorporation into productive processes for human pharmaceuticals would hopefully push the trend of marketed products and fulfil the increasing demands of the pharmacological industry. Keywords Gene cloning · Recombinant DNA technology · Vectors · Genes · Molecular medicine · Pharmaceuticals · Humulin · Post translational modifications · Clone · cDNA cloning · cDNA sequences · Genomic DNA libraries · cDNA library · Genomic cloning · Polymerase chain reaction · Recombinant DNA · Recombinant proteins · Glycosylation · Insect cell lines · Hamster cell lines · Hybridoma cell lines · Human cell lines · Recombinant DNA pharmaceuticals · Biopharming · Screening · Gene transfer · Gene therapy

M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_3,

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3 Recombinant DNA Pharmaceuticals

3.1 Prologue Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications (Manning et al., 1989). This has been accomplished successfully in microbial cells such as bacteria and yeasts. In early 1980s, the FDA approved the clinical use of recombinant human insulin from recombinant Escherichia coli (Humulin-US/Humuline-EU) for the treatment of diabetes, being the first recombinant pharmaceutical to enter the market. One hundred and fifty one recombinant pharmaceuticals have so far been approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) (Ferrer-Miralles et al., 2009). The versatility and scaling-up possibilities of the recombinant protein production opened up new commercial opportunities for pharmaceutical companies. Since the approval of recombinant insulin, other recombinant DNA drugs have been marketed in parallel with the development and improvement of several heterologous protein production systems. This has generated specific strains of many microbial species adapted to protein production, and has allowed the progressive incorporation of yeasts and eukaryotic systems for this purpose. Among the approved protein-based recombinant pharmaceuticals licensed up to January 2009 by the FDA and EMEA, 45 (29.8%) are obtained in E. coli, 28 (18.5%) in Saccharomyces cerevisiae, 17 (11.2%) in hybridoma cells, 1 in transgenic goat milk, 1 in insect cells and 59 (39%) in mammalian cells. Recombinant DNA (rDNA) technologies now offer a very potent set of technical platforms for the controlled and scalable production of polypeptides of interest by relatively inexpensive procedures. When massively expressed in host like bacteria, recombinant proteins often tend to misfold and accumulate as soluable and insoluable aggregates (de Marco et al., 2005. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional posttranslational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems developed for protein drug production.

3.2 Concept Genetic engineering and biomedical research have experienced such a revolutionary change since the past 30 years with the development of gene manipulation. Most biomolecules exist in low concentrations & as complex, mixed populations. This problem was solved in 1970 using the molecular biologist’s favourite bug, E. coli,

3.3

Tools of Recombinant DNA Technology

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a normally innocuous commensal occupant of the human gut. The basis of the term recombinant DNA refers to the recombining of different segments of DNA (Gluck et al., 1992). Constructing a recombinant DNA molecule involves cutting the DNA into fragments with restriction endonucleases and rejoining the fragments in novel arrangements with ligase. Propagating the molecule in a microorganism, or cloning, is necessary to increase the number of gene copies to facilitate detection of the gene of interest and to produce the protein it encodes (Black, 1989) in a desirable host. The purpose of these approaches is used to identify genes, to isolate the gene of interest, to amplify the gene if necessary, and to clone genes (Carroll, 1993). Fig. 3.1 General concept of recombinant DNA technology

3.3 Tools of Recombinant DNA Technology Recombinant DNA is a DNA molecule constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell. Most of the techniques of molecular genetics are either directly or indirectly dependent on molecules discovered in and isolated from bacteria. These molecules have functions in the everyday life of bacteria, but scientists have learned to exploit them as tools used for the manipulation and study of DNA. Any fragment of DNA can be cloned once it is introduced into a suitable vector for transforming a bacterial host. Cloning refers to the process of preparing multiple copies. cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5 - and 3 -untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. “Genomic cloning” refers to the production

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of a library of cloned DNAs representing the entire genome of a particular organism. From either of these types of libraries one can isolate a single cDNA or gene clone. In order to clone either cDNAs or copies of genes a vector is required to carry the cloned DNA. Once cloned, DNA fragments can be characterized by restriction enzyme mapping, by DNA sequencing, or by gene expression studies.

3.3.1 Common Enzymes Used in Recombinant DNA Technology The ability to manipulate DNA in vitro (outside the cell) depends entirely on the availability of purified enzymes that can cleave, modify and join the DNA molecule in specific ways. At present, no purely chemical method can achieve the ability to manipulate the DNA in vitro in a predictable way. Only enzymes are able to carry out the function of manipulating the DNA. Each enzyme has a vital role to play in the process of genetic engineering. The technique basically involves the use of several recently discovered enzymes (Table 3.1) that facilitate cutting and joining DNA to construct recombinant molecules, which can be transferred from one organism to another (Siddiqui, 1982). Table 3.1 Enzymes used in gene cloning Enzyme(s) Restriction endonucleases

Activity

Recognize specific nucleotide sequences and cleaves the DNA within or near to the recognition sequences Reverse Retrovirally encoded transcriptase RNA-dependent DNA (RT) polymerase RNase H Recognizes RNA-DNA duplexes and randomly cleaves the phosphodiester backbone of the RNA DNA polymerase Synthesis of DNA

Klenow DNA polymerase

DNA ligase Alkaline phosphatase

Comments Used to specifically recognize and cleave the double stranded DNA

Used to convert mRNA in to a complimentary DNA (cDNA) copy for the purpose of cloning cDNAs Used primarily to cleave the mRNA strand that is annealed to the first strand of cDNA generated by reverse transcription

Used during most procedures where DNA synthesis is required, also used in in vitro mutagenesis Proteolytic fragment of DNA Used to incorporate radioactive nucleotides polymerase that lacks the into restriction enzyme generated ends of 5 → 3 exonuclease activity DNA, also can be used in place of DNA polymerase Covalently attaches a free 5 Used in all procedures where to molecules of phosphate to a 3 hydroxyl DNA need to be covalently attached Removes phosphates from 5 Used to allow 5 ends to be subsequently radiolabeled with the phosphate of ATP in ends of DNA molecules the presence of polynucleotide kinase, also used to prevent self-ligation of restriction enzyme digested plasmids and lambda vectors.

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Table 3.1 (continued) Enzyme(s)

Activity

Comments

It is a bifunctional 5 -kinase/3 -phosphatase that aids in the repair of broken termini in RNA by converting 3 -PO4 /5 -OH ends into 3 -OH/5 -PO4 ends, which are then sealed by RNA ligase DNase I Randomly hydrolyzes the Is used in the identification of regions of phosphodiester bonds of DNA that are bound by protein and thereby double-stranded DNA protected from DNase I digestion, also used to identify transcriptionally active regions of chromatin since they are more susceptible to DNase I digestion S1 nuclease Exonuclease that recognizes Used to remove regions of single single stranded regions of strandedness in DNA or RNA-DNA DNA duplexes Exonuclease III Exonuclease that removes Used to generate deletions in DNA for nucleotides form the 3 end sequencing, or to map functional domains of DNAs of DNA duplexes Terminal DNA polymerase that requires Used to introduce homopolymeric (same transferase only a 3 -OH, lengthens 3 dNTP) tails onto the 3 ends of DNA duplexes, also used to introduce ends with any dNTP radiolabeled nucleotides on the 3 ends of DNA T3, T17 and SP6 Bacterial virus encoded RNA Used to synthesize RNA in vitro RNA polymerase, recognize polymerase specific nucleotide sequences for initiation of transcription Thermostable DNA Used in PCR Taq and Vent polymerases DNA polymerase

Polynucleotide kinase

Introduces phosphate of ATP to 5 ends of DNA

One vital type of molecule discovered in bacteria is the restriction endonuclease. These enzymes recognize specific base sequences within DNA molecules, and cleave specific phosphodiester bonds within that sequence. The key to the in vitro utilization of restriction endonucleases is their strict nucleotide sequence specificity. The different enzymes are identified by being given a name indicating the bacteria from which they were isolated, e.g. the enzymes EcoRI which recognizes the sequences, 5 -GAATTC-3 , was isolated from E. coli. One unique feature of restriction enzymes is that the nucleotide sequences they recognize are palindromic, i.e. they are the same sequences in the 5 → 3 direction of both strands. Some restriction endonucleases make staggered symmetrical cuts away from the center of their recognition site within the DNA duplex, some make symmetrical cuts in the middle of their recognition site while still others cleave the DNA at a distance from the recognition sequence (Fig. 3.1). Enzymes that make staggered cuts leave the resultant DNA with cohesive or sticky ends. Enzymes that cleave the DNA at the

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center of the recognition sequence leave blunt-ended fragments of DNA. It was also shown that an enormous variety of different restriction-modification systems, each with their own characteristic pair of nuclease and methylase for certain DNA recognition sequences, exist in different bacterial species. Any two pieces of DNA containing the same sequences within their sticky ends can anneal together and be covalently ligated together in the presence of DNA ligase. Any two blunt-ended fragments of DNA can be ligated together irrespective of the sequences at the ends of the duplexes. Other modifying enzymes (Table 3.1) includes phosphatases, kinases, single-strand specific nucleases, etc. that allow precise modifications to pieces of DNA to be made in vitro in order to add, remove or alter the structure of DNA. The RNA modifying enzymes – e.g. exonucleases, RNA ligase, reverse transcriptase. RNA is much more difficult to work with in vitro because the enzymes available are generally not as sophisticated as the set available which modify DNA. DNA Polymerases allow the synthesis of DNA from a pre-existing template in vitro, which can then be used for a variety of purposes, including sequence analysis by chain-termination methods. Recently, thermostable polymerases have become important, e.g. Taq DNA polymerase from Thermus aquaticus. This bacterium has evolved to grow in hot springs at temperatures which kill most other species. These enzymes allow the amplification of as little as one molecule of DNA into a large amount by means of repeated cycles of melting, primer annealing & extension by the enzyme which is not destroyed by the high temperatures used in this process. This is known as the polymerase chain reaction.

3.3.2 Vectors Vectors are segments of DNA replicated in living cells and engineered to allow the insertion of other DNA fragments. There are many types of vectors, including virus DNA, plasmid/virus hybrids, and artificial eukaryotic chromosomes (a shuttle vector is one that can replicate in 2 or more types of cells (e.g., E. coli and yeast)), but plasmids are the most common and easiest to understand. The insertion of DNA into a vector is done by cutting both the vector and DNA of interest with the same restriction enzyme. The sticky ends on each piece of DNA will be complementary to each other, due to the palindromic nature of the restriction enzyme recognition sites. Such DNA molecules in solution will transiently interact with each other, and join together via hydrogen bonds formed between sticky ends, forming a recombinant DNA molecule. If DNA ligase is present in the mixture with the two different fragments of DNA, any transiently-forming recombinant DNA molecules can be joined permanently to each other.

3.3.3 Hosts Once created, these recombinant DNA molecules are inserted into various hosts. The commonly used host is bacteria where by a process known as transformation these bacteria are successfully transformed with the DNA. They can be selected

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using the selectable marker. These transformed bacteria can then be grown up in great quantities, so that large amounts of the DNA can be recovered. This process, known as DNA cloning, is used for storage and amplification of recombinant DNA molecules. 1. Escherichia coli (E. coli) The enterobacterium E. coli is the first-choice microorganism for the production of recombinant proteins, and widely used for primarily cloning, genetic modification and small-scale production for research purposes (Sorensen and Mortensen, 2005). Recombinant proteins obtained in E. coli lack the posttranslational modifications (PTMs) which are present in most of eukaryotic proteins (Marston, 1996). PTMs play a crucial role in protein folding, processing, stability, final biological activity, tissue targeting, serum half-life and immunogenicity of the protein; therefore PMT deficient version might be insoluble, unstable or inactive. On the other hand, the frequencies with which the different codons appear in E. coli genes are different from those occurring in human genes, and this is directly related to the abundance of specific tRNAs. Therefore, genes that contain codons rare for E. coli may be inefficiently expressed by this organism and cause premature termination of protein synthesis or amino acid misincorporation, thus reducing the yield of expected protein versions. This problem can be solved either by sitedirected replacement of rare codons in the target gene by codons that are more frequently used in E. coli, or, alternatively, by the co-expression of the rare tRNAs (E. coli strains BL21 codon plus and Rosetta were designed for this purpose). In addition, initial methionine removal depends on the side chain of the penultimate amino acid of N-terminal in final recombinant proteins produced in E. coli although it can be efficiently removed using recombinant methionine aminopeptidase. Around 10% of full-length eukaryotic proteins tested in this system have been successfully produced in soluble form in E. coli. 2. Saccharomyces cerevisiae Production in yeast is usually approached when the target protein is not produced in a soluble form in the prokaryotic system or a specific PTM, essential for its biological activity, cannot be produced artificially on the purified product. Yeasts are as cost effective, fast and technically feasible as bacteria and high density cell cultures can also be reached in bioreactors (Branduardi et al., 2008). Even more, mutant strains that produce high amounts of heterologous protein are already available. Even though yeasts are able to perform many PTMs as O-linked glycosylation, phosphorylation, acetylation and acylation, the main pitfall of this expression system is related to N-linked glycosylation patterns which differ from higher eukaryotes, in which sugar side chains of high mannose content affect the serum half-life and immunogenicity of the final product. Although less studied than in bacteria, the production of recombinant proteins also triggers conformational stress responses and produced proteins fail sometimes to reach their native conformation.

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3. Insect Cell Lines Cultured insect cells are used as hosts for recombinant baculovirus infections. The production of a recombinant viral vector for gene expression is time-consuming, the cell growth is slow when compared with former expression systems, the cost of growth medium is high and each protein batch preparation has to be obtained from fresh cells since viral infection is lethal. PTMs are also an important limitation of this expression system because of the simple non-syalated N-linked glycosylation which is translated in a rapid clearance from human sera. 4. Hybridoma Cell Lines Hybridomas are fusion cells of murine origin (B-cells and myeloma tumour cells) that are able to express specific monoclonal antibodies against a determined antigen, thus possessing therapeutic potential. Clone selection may account for the progressive enrichment of cells displaying a glycosylation profile with reduced potency and undesirable immunogenic reaction since the human immune system recognizes mouse antibodies as foreign. 5. Hamster Cell Lines Most of the therapeutic proteins approved so far have been obtained using transgenic hamster cell lines, namely 49 in Chinese hamster ovary cells (CHO) and 1 in baby hamster kidney cells (BHK). The main advantage of this expression system is that cells can be adapted to grow in suspension in serum free media (SFM), protein-free and chemically defined media. This fact increases the biosafety of final products reducing risk of introducing prions of bovine spongiform encephalopathy (BSE) from bovine serum albumin and of infectious variant Creutzfeldt – Jakob disease (vCJD) from human serum albumin. In addition, recombinant products can be secreted into the chemical defined media, which simplifies both upstream and downstream purification process. PTMs in this expression system are almost the same as in human cell lines, although some concerns about comparability in the glycosylation pattern have arisen when comparing different batches of the same manufacturer product. Further development of chemically defined media and fine description of growth conditions would help to overcome this issue. 6. Human Cell Lines In the recent years, three therapeutic proteins produced in human cell lines have been approved, namely Dynepo-erithropoietin, Elaprase-irudonate-2-sulfatase and Replagal-alfa-galactosidase A. These products are fully glycosylated human proteins, so this expression system should be addressed when heavily glycosylation is needed. In general, recombinant biopharmaceuticals obtained from mammalian cells cover a wider spectrum of pathological conditions than those obtained from microbes, and the distribution of applications is less biased than when observing products from E. coli or S. cerevisae (Fig. 3.2).

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Fig. 3.2 Importance of post translational modifications for recombinant proteins

7. Transgenic Animals Transgenic animals (avian and mammals) have been successfully used for the production of recombinant proteins secreted into egg white and milk respectively. Protein production using transgenic farm animals supposes a great biotechnological challenge in terms of safety concerns such as transmission of infectious diseases (including viral and prion infections) or adverse allergenic, immunogenic and autoimmune responses. In 2006, ATryn was the first and so far single approved rDNA biopharmaceutical using transgenic animals and validated manufacturer technology platform. It contains human antithrombin (432 amino acids) with 15% glycosylated moieties and is secreted into the milk of transgenic goats. Another product obtained from the milk of transgenic rabbits (Rhucin) has been recently denied for its approval by the EMEA although more tests of repeated treatment are underway to try again its approval. Despite such limited progress, if pharmacovigilance after patient treatment does not reveal any adverse side effects, we might envisage, in the next years, an increase in the approval rate of recombinant protein products from transgenic animal origin. Recombinant DNA biopharmaceuticals obtained from bacterial, yeast or mammalian cell culture bioreactors are quite effective as therapeutic agents although production costs are relatively high. One way to address the economic-cost benefit hurdle is through the use of transgenic organisms to manufacture biopharmaceuticals. Biopharming would dramatically reduce the cost of recombinant therapeutic

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proteins not only in the initial construction of production facilities but also the scale-up process and the final recombinant protein yield. Nonetheless, the fact that regulatory guidelines are being developed as the same time that the establishment of protein production processes is creating uncertainty within biotechnological companies to fulfil drug administration requirements. 8. Transgenic Plants Transgenic plants have been used as recombinant protein producers for research and diagnostic uses due to the advantageous low cost of cultivation, high mass production, flexible scale-up, lack of human pathogens and addition of eukaryotic PTMs. The first recombinant protein product obtained from transgenic tobacco (Staub et al., 2000; Travis et al., 1985) was human growth hormone and since then, many other products have been obtained (including antibodies, the surface antigen of the Hepatitis-B-Virus, industrial enzymes and milk proteins). Again, the main disadvantage is related to the plant specific PMTs introduced in recombinant proteins which produce adverse immune responses. Moreover, the possibility to spread the proteins in open fields and the negative public perception of the transgenic plants precludes the use of plants as an attractive expression system of therapeutic proteins.

3.3.4 DNA Libraries DNA libraries are mixtures of different restriction enzyme-digested DNA molecules ligated into vectors. Libraries are constructed using the same general approach outlined above, except that instead of inserting one type of DNA fragment into the vector, there are thousands or even millions of different DNA fragments inserted into vector molecules. There are essentially two types of DNA libraries, based on the source of the DNA: Genomic DNA Libraries are made from genomic DNA. Genomic DNA molecules are very large, so they must be fragmented into small enough pieces to insert into vectors. This is typically done through digestion with one or more appropriate restriction endonucleases, mechanical shearing, or a combination of the two processes. The DNA is then ligated into the vector, which could be a plasmid, but is more often a cosmid or a viral chromosome. cDNA Libraries are made from cDNA, which are DNA copies of mRNA molecules. To make cDNA, mRNA is isolated from a tissue or whole organism, and DNA is copied from the mRNA template using an enzyme called reverse transcriptase. This enzyme works like a DNA polymerase, except that it uses RNA as a template instead of DNA. The resulting cDNA molecules are then engineered so that they have restriction enzyme recognition sites at each end of every molecule, which allows them to be digested and inserted into a vector as outlined previously. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently.

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The difference between these two libraries is the nature of the DNA found in the library. A cDNA library, because it is derived from mRNA molecules, will only contain DNA representing transcribed genes. A genomic library will also contain gene sequences, but will also contain a lot of DNA that is not genes, because genes typically make up only about 1–5% of the total genomic DNA. However, this type of library will contain regulatory DNA sequences (such as enhancers and promoter sequences) that would not be present in a cDNA library. Therefore, the choice of library type depends on what type of DNA sequence a researcher wishes to study.

3.3.5 Library Screening Plating out the phage is the first step of the screening process. Replicas are produced by placing absorbent paper, nitrocellulose or nylon membranes, over the agar plates in such a way that the pattern of plaques on the original plate is maintained. Each replica is then incubated with an appropriate nucleic acid probe that is labelled in some way (e.g. radiolabelling) so as to facilitate selection. If the probe is a radiolabelled fragment of DNA, the location of the positive clones may be detected by autoradiography. To find a specific fragment of DNA in a library, screening cDNA libraries can be done using antibodies against the protein product encoded by the gene of interest. Some vectors used to create libraries allow the inserted DNA to be transcribed after transformation into bacteria. These vectors have promoters built in, and since cDNA is copied from mRNA, it is capable of encoding protein. The transcripts would then be translated into protein in the bacteria. Bacteria transformed with the library can be grown in duplicate copies on plates and on filters in a way that individual bacterial colonies are produced, with each colony originating from a single bacterial cell, so all bacteria in a colony would contain the same recombinant DNA molecule, and therefore produce the same protein. Many thousands of colonies can be grown on a series of filters, and the bacteria lysed (broken open), liberating the protein, which would then adhere to the filter. Filters can be exposed to antibodies that are labeled in some way (usually with a fluorescent or radioactive tag); the antibody to the protein of interest will bind only to the colony that contained the recombinant DNA molecule encoding that protein. Once the colony is identified, the duplicate colony from a bacterial plate can be picked, and grown up to produce more of that DNA. This method doesn t work well with genomic libraries, because most recombinant clones in a genomic library do not encode protein. Another way to screen a library is with a piece of DNA similar to the DNA of interest, such as a similar gene from a different organism, for example. This strategy takes advantage of the ability of DNA to denature and renature. In this case, transformed bacterial colonies are grown in duplicate on plates and filters. The bacteria on the filters are lysed, and the DNA is denatured by treating with an alkaline solution. The filters are then exposed to a solution containing a singlestranded DNA “probe”, labeled with a radioactive or chemical tag, under conditions that promote renaturation. If the probe DNA is in excess, sequences strongly related

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to the probe will renature with the probe, and can be detected by exposure to X-ray film. As with the antibody approach, the corresponding colony on the plates can be picked and isolated. All of the bacteria in this colony descended from a single bacterial cell, and therefore they all contain the same genetic complement. This means that the plasmid (or whichever vector was used) in every bacterial cell in the colony will contain the same insert. Once isolated, a bacterial colony can be inoculated into bacterial growth medium, which provides the bacteria with the necessary nutrients for fast reproduction. In a matter of hours, the total number of bacterial cells can be increased over one billionfold. The plasmid (or other vector) DNA can then be isolated from the bacteria, producing a sizable yield. In this way, it is possible to amplify the amount of a specific sequence of DNA. Bacteria from the growth culture can also be stored frozen at –70◦ C, and will remain viable for many years. Therefore, once a particular clone from a library is selected, it can be stored for years without having to rescreen the library for that sequence.

3.3.6 Polymerase Chain Reaction In the late 1980s, an alternative technique was developed by which DNA can be amplified many times over. This technique does not require the use of living bacteria or other cells, and is essentially a DNA replication reaction done in a test tube. We will discuss this technique in the chapter on PCR. PCR can be used in the analysis of disease genes (Table 3.2) by being able to amplify detectable amounts of specific fragments of DNA. The amplified fragments from disease genes may be larger, due to insertions, or smaller, due to deletions. The dramatic amplification of DNA by PCR allows the analysis of disease genes in extremely small samples of DNA. For example, only a small number of fetal cells need be extracted from amniotic fluid in order to analyze for the presence of specific disease genes. Additionally, single point mutations can be detected by modified PCR techniques such as the ligase chain reaction (LCR) and PCR-singlestrand conformational polymorphisms (PCR-SSCP) analysis. The PCR technique also can be used to identify the level of expression of genes in extremely small samples of material, e.g. tissues or cells from the body. This technique is termed reverse transcription-PCR (RT-PCR). Many inherited disorders are due to single nucleotide changes within critical regions of the affected gene (e.g. sickle cell anemia). Many inherited disorders (Table 3.2) are due to single nucleotide changes within critical regions of the affected gene (e.g. sickle cell anemia). The PCR-SSCP technique can detect single mutations in genes due to the altered conformation mobility of the single strands of DNA within an electrophoresis gel harboring the mutation relative to the wild-type strands that do not. Specific PCR primers are made that span the sequences of a given disease gene where a mutation is known to exist and the region amplified by PCR. The same region of the wild-type gene is PCR amplified. The two strands of wild-type PCR product will migrate differently than the two

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strands of mutant PCR product. Even single point mutations lead to the strands of amplified DNA existing in different conformations which alter their mobility when subjected to electrophoresis in non-denaturing gels. The most common techniques utilized in gene therapy studies is the introduction of the corrected gene into bone marrow cells, skin fibroblasts or hepatocytes. The vectors most commonly utilized are derived from retroviruses and utilize only the transcriptional promoter regions of these viruses (the LTRs) to drive expression of the gene of interest. The advantage of retroviral-based vector systems is that expression occurs in most cell types. A number of human inherited disorders have been corrected in cultured cells and several diseases (e.g. malignant melanoma and severe combined immunodeficiency disease, SCID) are currently being treated by gene therapy techniques indicating that gene therapy is likely to be a powerful therapeutic technique against a host of diseases in coming years. The technique of PCR has also revolutionized forensic science, because it allows amplification of DNA from minute samples such as blood droplets, which then allows DNA fingerprints to be performed.

Table 3.2 Diseases and disorders detected by PCR and their related gene Disease

Affected gene

Adenosine deaminase deficiency Lesch-Nyhan syndrome

Adenosine deaminase (ADA) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) α-1-Antitrypsin Cystic fibrosis transmembrane conductance (CFTR) protein β-Galactosidase Glucocerebrosidase Hexosaminidase A and B Hexosaminidase A LDL receptor Glucose-6-phosphate dehydrogenase

α-1-Antitrypsin deficiency Cystic fibrosis Fabry disease Gaucher’s disease Sandhoff-Jatzkewitz disease Tay-Sachs disease Familial hypercholesterolemia (FH) Glucose-6-phosphate dehydrogenase deficiency Maple syrup urine disease Phenulketonuria (PKU) Ornithine transcarbamylase deficiency Retinoblastoma (Rb) Sickle-cell anemia β-Thalassemia Hemophilia A Hemophilia B von Wilebrand disease

α-keto acid decarboxylase Phenylalanine Ornithine transcarbamylase Rb gene product Point mutation in globin gene resulting in improper folding of protein Mutations in β-globin gene that result in loss of synthesis of protein Factor VIII Factor IX von Wilebrand factor (vWF)

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3.3.7 Analysis of Cloned DNA Sequences Once DNA has been cloned and amplified, it needs to be analyzed and characterized. Two common ways to do this are restriction enzyme mapping and DNA sequencing, both of which rely upon a technique known as gel electrophoresis. Restriction enzyme mapping entails digesting the DNA of interest with various restriction enzymes, singly and in pairs. By running the various reactions on agarose gels, it is possible to determine the sizes of the digested fragments (by comparing to DNA standard fragments of known size). This in turn allows mapping of the different restriction enzyme recognition sites relative to each other. Sometimes, it is necessary to visualize only a few restriction fragments out of a large set of fragments; a technique known as Southern blotting is used. The analysis of cloned cDNAs and genes involves a number of techniques. The initial characterization usually involves mapping of the number and location of different restriction enzyme sites. This information is useful for DNA sequencing since it provides a means to digest the clone into specific fragments for sub-cloning, a process involving the cloning of fragments of a particular cloned DNA. Once the DNA is fully characterized cDNA clones can be used to produce RNA in vitro and the RNA translated in vitro to characterize the protein. Clones of cDNAs also can be used as probes to analyze the structure of a gene by Southern blotting or to analyze the size of the RNA and pattern of its expression by Northern blotting. Northern blotting is also a useful tool in the analysis of the exon-intron organization of gene clones since only fragments of a gene that contain exons will hybridize to the RNA on the blot. Western blotting involves the analysis of proteins following attachment to a solid support. The proteins are separated by size SDS-PAGE and electrophoretically transferred to nitrocellulose or nylon filters. The filter is then probed with antibodies raised against a particular protein. Ultimately, to properly study and utilize a piece of DNA, we need to know its sequence of bases. This can be done using a technique called DNA sequencing, which utilizes a modified DNA replication reaction. Sequencing of DNA can be accomplished by either chemical or enzymatic means. The original technique for sequencing, Maxam and Gilbert sequencing, relies on the nucleotide-specific chemical cleavage of DNA and is not routinely used any more. The enzymatic technique, Sanger sequencing, involves the use of dideoxynucleotides (2 ,3 -dideoxy) that terminate DNA synthesis and is, therefore, also called dideoxy chain termination sequencing.

3.3.8 Expression of Cloned Genes The main reason we clone and characterize genes is to produce the recombinant protein. This is done for a variety of reasons. Sometimes, the protein is a product that will be harvested for commercial sale by a biotechnology or pharmaceutical company. Production of the protein can also be used in an indirect way to understand its function. By expressing the gene in cells or an organism (for example, expressing a

3.4

Therapeutic Applications of Recombinant DNA Derived Pharmaceuticals

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gene at a time or in a place that it wouldn’t normally be expressed), it may be possible to observe the effect that expression has on the organism, and infer the function from that. Expression of a gene employs a class of vector known as an expression vector. These vectors have promoters (often from viral genes) engineered into the vector near the multiple cloning site. Any gene inserted into the multiple cloning site therefore comes under the control of the promoter, allowing expression of the gene under appropriate conditions. Expression may be in vivo (in a living cell or organism) or in vitro (in a test tube using cell-free transcription and translation systems that are available commercially, allowing production of the protein relatively purely).

3.4 Therapeutic Applications of Recombinant DNA Derived Pharmaceuticals Successful development of recombinant DNA-derived pharmaceuticals, a new class of therapeutic agents, is determined by a variety of factors affecting the selection and positioning of the compound under development. Technical development of recombinant DNA technology puts genetics in the mainstream of medicine. Techniques for gene transfer have been improved to introduce synthetic sequences into the cellular genomes. High quality of recombinant products covering monoclonal antibodies, vaccines thrombolytics have been developed by this techniques. Candidate cells and culture systems have been screened for their capacity to synthesise the proper recombinant molecules.

3.4.1 Recombinant Therapeutic Proteins Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical application (Manning et al., 1989). Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development. Substitution therapy with recombinant DNA derived human proteins is in therapeutic application to replace their counterparts from native source by human pharmacologically active proteins which cannot be isolated from their natural source. For recombinant DNA derived proteins where the mode of action is known short development times frames can be expected allowing an early return on investment. Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties. These properties pose difficult stability problems. After more than half a century of treating diabetics with animal insulin, recombinant DNA technologies and advanced protein chemistry made human insulin preparations available in the early 1980s (Vajo et al., 2001). Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology (Johnson, 1983). Large quantities of biosynthetic human proinsulin have recently become available through recombinant DNA technology (Revers et al., 1984). A chimeric bovine GH (amino acids Met-Asp-Gln-greater

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than 1-23) and human GH (hGH) (amino acids 24-191) plasmid was constructed and expressed in E. coli (Binder et al., 1989). Human follicle-stimulating hormone (FSH) (Loumaye et al., 1995) and tissue-type plasminogen activator (Tiefenbrunn et al., 1985) are now produced in vitro by recombinant DNA technology. Approved therapeutic protein-based products from E. coli include hormones (human insulin and insulin analogues, calcitonin, parathyroid hormone, human growth hormone, glucagons, somatropin and insulin growth factor 1), interferons (alfa-1, alfa 2a, alfa-2b and gamma-1b), interleukins 11 and 2, light and heavy chains raised against vascular endothelial growth factor-A, tumor necrosis factor alpha, cholera B subunit protein, B-type natriuretic peptide, granulocyte colony stimulating factor and plasminogen activator. Noteworthy, most of the recombinant pharmaceuticals produced in E. coli are addressed for the treatment of infectious diseases or endocrine, nutritional and metabolic disorder disease groups (Werner, 1990; Redwan et al., 2008). The approved protein products produced in yeast are obtained exclusively in S. cerevisiae and correspond to hormones (insulin, insulin analogues, non glycosylated human growth hormone somatotropin, glucagon), vaccines (hepatitis B virus surface antigen) and virus-like particles (VLPs) of the major capsid protein L1 of human papillomavirus type 6, 11, 16 and 18, urate oxidase from Aspergillus flavus, granulocyte-macrophage colony stimulating factor, albumin, hirudin of Hirudo medicinalis and human platelets derived growth factor. As in the case of E. coli, most of the recombinant pharmaceuticals from yeast are addressed to either infectious diseases or endocrine, nutritional and metabolic disorders, being these therapeutic areas the most covered by microbial products. Interestingly, several yeast species other than S. cerevisiae are being explored as sources of biopharmaceuticals and other proteins of biomedical interest (Porro et al., 2005). In addition, current metabolic engineering approaches and optimization of process procedures (Graf et al., 2008; Mattanovich et al., 2004) are dramatically expanding the potential of yeast species for improved production of recombinant proteins. There is only one approved biopharmaceutical product containing recombinant proteins from infected insect cell line Hi Five, Cervarix, consisting of recombinant papillomavirus C-terminal truncated major capsid protein L1 types 16 and 18. Nonetheless, this expression system has been extensively used in structural studies since correctly folded eukaryotic proteins can be obtained in a secreted form in serum free media which enormously simplifies protein capture in purification protocols. Genetic engineering has been applied to obtain humanized monoclonal antibodies using either recombinant mammalian cells producing chimeric antibodies or genetically modified mice to produce human-like antibodies. One such product, Remicade, which binds tumour necrosis factor-alpha, is a pharmaceutical blockbuster used in the treatment of Crohn’s disease. Most of the therapeutic proteins approved so far have been obtained using transgenic hamster cell lines, namely 49 in Chinese hamster ovary cells (CHO) and 1 in baby hamster kidney cells (BHK). The main advantage of this expression system is that cells can be adapted to grow in suspension in serum free media (SFM), protein-free and chemically defined media.

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This fact increases the biosafety of final products reducing risk of introducing prions of bovine spongiform encephalopathy (BSE) from bovine serum albumin and of infectious variant Creutzfeldt – Jakob disease (vCJD) from human serum albumin. In addition, recombinant products can be secreted into the chemical defined media, which simplifies both upstream and downstream purification process. PTMs in this expression system are almost the same as in human cell lines, although some concerns about comparability in the glycosylation pattern have arisen when comparing different batches of the same manufacturer product. Further development of chemically defined media and fine description of growth conditions would help to overcome this issue. Recombinant structural proteins include hepatitis B virus vaccine and CD4 protein, and recombinant modifier proteins include tissue plasminogen activator and superoxide dismutase (agents that split or splice organic molecules). In the future, gene defects associated with genetic diseases will be unraveled, leading to the production of new therapeutic agents designed to counteract or actually reverse those defects. Recombinant protein drugs will be further tailored to enhance their activity and specificity. These advances are so novel and momentous that patent protection has been extended not only to recombinant drugs but to the recombinant microorganisms in which they are manufactured. In cloning genes, investigators directly use the protein designs that occur naturally. To use recombinant DNA technology to functionally analyze mutations introduced into cloned eukaryotic genes, a rapid procedure is necessary to assay the steps along the gene expression pathway. If one assays shortly after its introduction into mammalian cells, it can be shown that this recombinant plasmid programs the synthesis of correctly spliced and polyadenylylated insulin mRNA that functions in the synthesis and secretion of rat proinsulin. This system permits rapid analysis of cloned in vitro – engineered mutations and the programming of eukaryotic cells to manufacture proteins that they normally do not synthesize. Basic research will soon lead to the engineering of novel proteins with specified functions.

3.4.2 Recombinant DNA Technology and Medicine Recombinant DNA technology has already had a major impact on our understanding of microbiology, cell biology, developmental biology (Dawid, and Wahli, 1979) and genetic diseases and it will certainly have extensive applications in laboratory medicine. In the nearest future, high biotechnologies should provide a great quantity of recombinant products, covering a high variety of diseases (clotting factors, cytokines, hormones, monoclonal antibodies, vaccines, thrombolytics). Besides, genetic engineering offers new therapeutic outlets: transgenic animals, genic therapy, antisense technology (Descamps, 1994). In case of a single gene defect a number of appropriate gene probes are available for prenatal diagnosis. Knowledge of the genetic disorders enables in some cases early onset of therapy or the option for abortion. However, gene technology which enables the diagnosis should not be viewed from an ethical point of view but rather the action taken when diagnostic results are available.

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Gene therapy for a single gene defect still is at the early stage of development. Only few patients have been treated in various indications. Difficult to be overcome are the low frequency and unspecific integration of inserted DNA into the chromosome, lack of sufficient transcription control and short half-lives of the integrated gene. From an ethical perspective gene therapy complies with the therapeutic concept of medicine. Antisense oligonucleotides are under clinical development for blockage of the synthesis of oncogenes and viral proteins. Stability of oligonucleotides as well as selectivity for specific cells will have to be overcome for broader application. Its therapeutic application is in accordance with the ethical principles of medicine. Recombinant DNA procedures have now been applied to the problem of the identification of molecular defects in man that account for heritable diseases, somatic mutations associated with neoplasia, and acquired infectious diseases. Genetic tests will predict common diseases as well as many rare ones and touch the lives of most people (Holtzman, 1988). Thus recombinant DNA technology has rapidly expanded our ability to diagnose disease (Berg et al., 1974). Substantial advances in the simplification of procedures for diagnostic purposes have been made, and the informed physician has gained in diagnostic accuracy as a consequence of these developments. The wide application of recombinant DNA diagnostics will depend on simplicity, speed of results, and cost containment. As yet recombinant DNA technology does not appear to have widespread diagnostic application in pathology. However, it does have a useful role to play in specific circumstances in at least three main areas: 1. It can provide precise diagnostic information about genetic diseases, allowing appropriate counselling, and indicating future directions for research on therapeutic intervention, e.g. gene therapy 2. Micro-organisms can be identified more sensitively and specifically, in fresh or fixed tissue samples, and their genomes can be analysed in fine detail, providing information relevant to the aetiology, epidemiology and pathogenesis of many diseases 3. In tumour pathology the main application so far has been to resolve diagnostic problems associated with leukaemias and lymphomas, when other diagnostic procedures have been inconclusive. Specific chromosomal translocations, involving recognized genes, are particularly amenable to diagnosis by these means. Diagnostic applications to solid tumours are yet to be identified, although significant insights into tumorigenesis have been obtained, and these may ultimately lead to the development of useful markers for prognostic and therapeutic purposes. The techniques of restriction endonuclease analysis of DNA, nucleic acid hybridization after electrophoretic separation of nucleic acid fragments, and molecular cloning of bacterial, viral, and human genes are already being used in epidemiologic studies and the prenatal diagnosis of certain genetic diseases, such as sickle cell anemia and the thalassemias. New insights into genes that may be

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involved in human cancer are being developed and may lead to improved methods for diagnosis and classification of tumors. Techniques for gene transfer have been improved to introduce synthetic sequences into the cellular genomes. Candidate cells and culture systems have been screened for their capacity to synthesise the proper recombinant molecules. In the nearest future, high biotechnologies should provide a great quantity of recombinant products, covering a high variety of diseases (clotting factors, cytokines, hormones, monoclonal antibodies, vaccines, thrombolytics). This tendency has been largely initiated, and is accompanied by the quick development of basic sciences such as glycobiology and molecular biology, together with the development of a specific regulatory environment. Besides, genetic engineering offers new therapeutic outlets: transgenic animals, gene therapy, antisense technology (Descamps, 1994). Recombinant DNA drug products have been developed that represent the communicator, structural, and modifier classes of proteins. Recombinant communicator proteins include interferons alfa-2a and alfa-2b and granulocyte-macrophage colony-stimulating factor (immune system modulators) epidermal growth factor and erythropoietin (tissue repair promoters) and human insulin, growth hormone, and atrial peptide (metabolism modulators). Regulation of expression of specific genes has impacted on understanding of cancer development and immunopathologic diseases. An even greater impact has been the production of new therapeutic molecules. Cytokines have been produced by recombinant DNA technology and are established as effective therapeutic modalities for cancer and infectious diseases. High quality pharmaceutical molecules have been produced which can substantially regulate cell function. Interferons typify this progress and are now licensed for both viral and neoplastic disease in more than 43 countries around the world. Parallel development of monoclonal antibody (MAb) and recombinant DNA technologies has enabled the design and production of several potent biologics that can specifically block the deleterious effects of TNFα. Two MAbs (infliximab and adalimumab), as well as a receptor fusion protein (etanercept), have thus far proven safe and efficacious in pivotal rheumatoid arthritis clinical trials. In a recent study recombinant proteins were reported to be used as specific molecular markers to differentiate between acute and chronic infections. Holec-Gasior and his colleagues (2009) reported GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in humans with acute Toxoplasmosis. These evolving technologies provide the foundation from which future biotherapies can be derived as targets are identified and validated in diverse disease states.

3.5 Conclusion Overcoming the biological and methodological obstacles posed by cell factories to the production or rDNA pharmaceuticals is a main challenge in the further development of protein-based molecular medicine. Recombinant DNA technologies might have exhausted conventional cell factories and new production systems need to

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be deeply explored and incorporated into the production pipeline. Interestingly, a plateau in the rate of rDNA drug approval during the last 2–3 years is becoming perceivable, irrespective of the production system. Although it might be observed as a transient event, this fact seems instead to indicate that the current production systems could be near to the exhaustion regarding their ability to hold the production of complex proteins, protein complexes or the so-called difficult-to-express heterologous proteins (Jana and Deb, 2005). Desirably, recent insights about system’s biology of recombinant cells and hosts, and specially, arising novel concepts on recombinant protein quality (Gonzalez-Montalban, 2007; de Marco, 2007) and host stress responses would enlarge the possibilities for metabolic and process engineering aiming to the economically feasible production of new, more complex drugs. Indeed, pushed by fast advances in molecular medicine the pharmaceutical industry is urgently demanding improved production systems and novel and cheaper drugs. Their incorporation into productive processes for human pharmaceuticals would hopefully push the trend of marketed products and fulfil the increasing demands of the pharmacological industry.

References Berg, P., Baltimore, D., Boyer, H.W., 1974, Potential biohazards of recombinant DNA molecules. Science 185:303. Binder, L., Vogel, T., Hadary, D., 1989, Chimeric bovine-human growth hormone prepared by recombinant DNA technology: binding properties and biological activity. Mol Endocrinol 3:923–930. Black, W.J., 1989, Drug products of recombinant DNA technology American. J HospPharm 46:1834–1844. Branduardi, P., Smeraldi, C., Porro, D., 2008, Metabolically engineered yeasts: ‘potential’ industrial applications. J Mol Microbiol Biotechnol 15:31–40. Carroll, W.L., 1993, Introduction to recombinant-DNA technology American. J Clin Nutr 58:249S–258S. Dawid, I.B., Wahli, W., 1979, Application of recombinant DNA technology to questions of developmental biology: a review. Dev Biol 69:305–328. de Marco, A., 2007, Protocol for preparing proteins with improved solubility by co-expressing with molecular chaperones in Escherichia coli. Nat Protoc 2:2632–2639. de Marco, A., Schroedel, A., 2005, Characterization of the aggregates formed during recombinant protein expression in bacteria. BMC Biochem 6:10. doi: 10.1186/1471-2091-6-10. Descamps, J.L., 1994, Therapeutic applications of genetic engineering. Ann Pharm Fr 52: 294–302. Ferrer-Miralles, N., Domingo-Espín, J., Corchero, J.L., Vázquez, E., Villaverde, A., 2009, Microbial factories for recombinant pharmaceuticals. Microb Cell Fact 8:17, doi:10.1186/1475-2859-8–17. Gluck, S.L., Nelson, R.D., Lee, B.S., 1992, Introduction to the principles and methods of recombinant DNA technology. Semin Nephrol 12:467–494. Gonzalez-Montalban, N., 2007, Recombinant protein solubility-does more mean better. Nat Biotechnol 25:718–720. Graf, A., Gasser, B., Dragosits, M., Sauer, M., Leparc, G.G., Tüchler, T., Kreil, D.P., Mattanovich, D., 2008, Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays. BMC Genomics 9:390.

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Holec-Gasior, L., Kur, J., Hiszczyńska-Sawicka, E., 2009, GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in humans with acute toxoplasmosis. Clin Vaccine Immunol 16:510–514. Holtzman, N.A., 1988, Recombinant DNA technology, genetic tests, and public policy. Am J Hum Genet 42:624–632. Jana, S., Deb, J.K., 2005, Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298. Johnson, I.S., 1983, Human insulin from recombinant DNA technology. Science 219:632–637. Lomedico, P.T., 1982, Use of recombinant DNA technology to program eukaryotic cells to synthesize rat proinsulin: a rapid expression assay for cloned genes. PNAS 79:5798–5802. Loumaye, E., Campbell, R., Salat-Baroux, J., 1995, Human follicle-stimulating hormone produced by recombinant DNA technology: a review for clinicians. Hum Reprod Update 1:188–199. Manning, M.C., Patel, K., Borchardt, R.T., 1989, Stability of protein pharmaceuticals. Pharm Res 6:903–918. Marston, F.A., 1986, The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J 240:1–12. Mattanovich, D., Gasser, B., Hohenblum, H., Sauer, M., 2004, Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135. Porro, D., Sauer, M., Branduardi, P., Mattanovich, D., 2005, Recombinant protein production in yeasts. Mol Biotechnol 31:245–259. Redwan, e., Matar, S.M., El-Aziz, G.A., Serour, E.A., 2008, Synthesis of the human insulin gene: protein expression, scaling up and bioactivity. Prep Biochem Biotechnol 38:24–39. Revers, R.R., Henry, R., Schmeiser, L., Kolterman, O., Cohen, R., Bergenstal, R., Polonsky, K., Jaspan, J., Rubenstein, A., Frank, B., 1984, The effects of biosynthetic human proinsulin on carbohydrate metabolism. Diabetes 33:762–770. Siddiqui, M.A., 1982, Recombinant DNA technology and its application to developmental biology. J Craniofac Genet Dev Biol 2:75–92. Sorensen, H.P., Mortensen, K.K., 2005, Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4:1. Staub, J.M., Garcia, B., Graves, J., Hajdukiewicz, P.T., Hunter, P., Nehra, N., Paradkar, V., Schlittler, M., Carroll, J.A., Spatola, L., Ward, D., Ye, G., Russell, D.A., 2000, High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338. Tiefenbrunn, A.J., Robinson, A.K., Kurnik, P.B., Ludbrook, P.A., Sobel, B.E., 1985, Clinical pharmacology in patients with evolving myocardial infarction of tissue-type plasminogen activator produced by recombinant DNA technology. Circulation 71:110–116. Travis, J., Owen, M., George, P., 1985, Isolation and properties of recombinant DNA produced variants of human alpha 1-proteinase inhibitor. J Biol Chem 260:4384–4389. Vajo, Z., Fawcett, J., Duckworth, W.C., 2001, Recombinant DNA technology in the treatment of diabetes: insulin analogs. Endocr Rev 22:706–717. Werner, R.G., Pommer, C.H., 1990, Successful development of recombinant DNA-derived pharmaceuticals. Arzneimittelforschung 40:1274–1283.

Chapter 4

Aptamers: In Vitro DNA Selection

Abstract Aptamers are a new class of therapeutic and diagnostic reagents identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX). This method is an in vitro method of selection of DNA or RNA sequences, involving several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. These range from inhibition of receptors and enzymes to the identification of small molecules in sensor applications, and from the development of targeted therapeutic to the design of novel diagnostic and imaging agents. Furthermore, aptamers have been designed for targets that cover a wide range of diseases, from HIV to tropical diseases, cancer and inflammation. Their easy development and flexibility of use and manipulation, offers further potential as effective diagnostic tools. Keywords Aptamers · Pathogens · Therapeutics · Amplification · DNA selection · RNA aptamers · Oligomers · SELEX · Oligonucleotide library · MonoLEX · Combinatorial library · DNA aptamers

4.1 Prologue Effective catalysis involves the specific recognition of a target molecule with high affinity. Although many catalytic functions have evolved naturally, mostly in proteins, the need for new catalytic functions is growing; for example to convert industrial byproducts to useful products. To develop chemicals that recognize new targets such as industrial byproducts, combinatorial molecular biology are now-a-days being used. Aptamers are such new products that form unique structures to meet the requirements for more selective binding and sensitive recognition modules for increasingly sensitive detection methods that are required in natural sciences and medicine for the detection and quantization of molecules. M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_4,

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4 Aptamers: In Vitro DNA Selection

4.2 Concept Aptamers are short, single-stranded nucleic acid (DNA or RNA) oligomers that have been selected in vitro to bind with high affinity and specifically to a certain molecular target (Lou, 2009; Ellington and Szostok, 1990). They are 50–100 base pair in length that bind to proteins bind with Kds (equilibrium constant) in the range of 1 pM–1 nM similar to monoclonal antibodies. The molecular target may be amino acids, drugs, proteins, nucleic acids small organic compounds, and even entire organisms (Joshi et al., 2009; Mairal, 2008). The term “aptamer” is derived from Latin aptus (= fitting) and Greek meros (= part) and was chosen to illustrate “lock and key model” between aptamers and their binding partners. Due to the multitude of structures which can be formed by aptamers, they are able to recognize virtually all classes of substrates and bind them perfectly fitting in analogy to antigen-antibody interactions. In such a way, aptamers were developed for atoms and small molecules, for amino acids, peptides, polysaccharides, and proteins, but also for complex targets like viruses and protozoa. The selected sequences have the ability to recognize specific ligands by forming binding pockets and can bind to nucleic acids (Le Tinevez et al., 1998; Pileur et al., 2003), proteins (Bock et al., 1992; Lupold et al., 2002) or small organic compounds (Berens et al., 2001; Burgstaller and Famulok, 1996).The aptamer-target interaction takes place via structural compatibilities of the two binding partners, via electrostatic interactions, like Van der Waals interactions, ionic or dipole forces, hydrogen bonds, or stacking interactions. These nucleic acid ligands bind to nucleic acid, proteins. Aptamer recognition affinity and specificity is comparable to those of monoclonal antibodies and aptamers can be selected to recognize and bind a wide range of targets, including toxic compounds and inherently non-immunogenic molecules that antibodies cannot be raised against. Furthermore, as aptamers are 10–100 times smaller than antibodies, they are expected to achieve higher tumour penetration than their counterparts (Jayasena, 1999). Aptamers have many potential uses in intracellular processes studies, medicine and technology. In addition to the genetic information encoded by nucleic acids they also function as highly specific affinity ligands by molecular interaction based on the three dimensional folding pattern. The three dimensional complex shape of a single stranded oligonucleotide is primarily due to the base composition led intramolecular hybridization that initiates folding to a particular molecular shape. This molecular shape assists in binding through shape specific recognition to it targets leading to considerable three dimensional structure stability and thus the high degree of affinity. Natural examples of molecular shape recognition interactions of nucleic acids with proteins are tRNA, ribozymes, DNA binding proteins and DNAzymes. DNA aptamers are heat and protease resistant without stabilizing modifications. Due to chemical synthesis, aptamer production can easily be scaled up (Jellinck et al., 1993). Once identified, aptamers can be reliably synthesized by automated methods. During synthesis, aptamers can be easily modified to facilitate further modifications such as attachment points for enzymes or a variety of other reagents, and the introduction of modified bases for nuclease resistance (Brody and Gold,

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Selection of Aptamers

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2000; Eaton et al., 1997; Gewirtz, 1999). Furthermore, aptamers can be stored until needed and are resistant to denaturation and degradation when lyophilised. During the selection process aptamers with the highest affinity to a target structure are isolated from a pool of oligonucleotides with random positions for nucleic acids, which, depending on the length of the aptamers, contain up to 1015 different sequence variants, representing a significantly larger pool of variants than antibodies do. In addition to these evident benefits, aptamers exhibit some drawbacks, including their reduced resistance to nucleases and the problem of delivery and clearance in therapeutic application. First attempts to stabilize aptamers were promising (Ruckman et al., 1998; Morris et al., 1998).

4.3 Selection of Aptamers Theoretically it is possible to select aptamers virtually against any molecular target. Aptamers have been selected for small molecules, peptides, proteins as well as viruses and bacteria. The aptamers are selected by incubating the target molecule in a large pool of oligonucleotide (usually 40–60 mers); the pool size of the oligonucleotide is from 1,010 to 1,020. The large pool size of the oligonucleotide ensures the selection and isolation of the specific aptamer. The structural and informational complexity of the oligonucleotide pool and its functional activity is an interesting and active area to develop an algorithm based development of nucleic acid ligands. Aptamers can distinguish between closely related but non-identical members of a protein family, or between different functional or conformational states of the same protein. In a striking example of specificity, an aptamer to the small molecule theophylline (1,3-dimethylxanthine) binds with 10,000-fold lower affinity to caffeine (1,3,7-trimethylxanthine) that differs from theophylline by a single methyl group. The SELEX method (systematic evolution of ligands by exponential enrichment) (Ellington and Szotok, 1990) is an oligonucleotide-based combinatorial library approach that has been extensively used to isolate high-affinity ligands (called aptamers) for a wide variety of proteins and small molecules (Jellinek et al., 1993). This is a method in which single stranded oligonucleotides are selected from a wide variety of sequences, based on their interaction with a target molecule. Using this process, it is possible to develop new aptamers in as little as 2 weeks. Starting point of each SELEX process is a synthetic random DNA oligonucleotide library (RNA, ssDNA or modified oligonucleotide molecules). Normally, 1015 different molecules are employed. Regarding this multitude of possible structures, it is very likely to select at least one binding partner (aptamer) for virtually each target. In order to amplify these target binding structures, the oligonucleotide library is subjected to an adequate selection process in the presence of the target molecules. During this process, relevant nucleic acid molecules are separated from non-relevant ones. Target binding aptamers subsequently are amplified and repeatedly subjected to interactive selection processes until relevant nucleic acid molecules dominate the oligonucleotide pool. The enriched aptamer pool is cloned and several individual aptamers can be sequenced and characterized. As a result, the

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Fig. 4.1 The SELEX method is an oligonucleotide-based combinatorial library approach to isolate high-affinity ligands called aptamers for a wide variety of proteins and small molecules

selected aptamers are ready to be ultilized according to the given task (Fig. 4.1). However, this procedure requires repetitive cycles and is therefore very timeconsuming. The in vitro selection of RNA and DNA ligands against specific targets obeys the same rules as natural selection. For this purpose a partial randomized synthetic DNA template is constructed containing a random inner region that is flanked on both sides by constant sequences. The random sequence classically consists of 15–75 random positions where all four bases are incorporated with equal probabilities. This pool containing 1012 –1015 different sequences can be either directly used for selection or first transcribed to RNA using T7 RNA polymerase. In this case a T7 promoter site needs to be placed on the 5 site of the DNA template. The random DNA/RNA pool is exposed to the protein target and the best fitting molecules in the selection pool are culled and amplified. The procedure will be repeated with increasing stringency until the previous random pool is purified to a few molecules with the desired binding properties. The final pool is cloned into a bacterial vector and individual colonies are sequenced (Fig. 4.2). The previous random regions are aligned and searched for consensus

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Selection of Aptamers

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Fig. 4.2 The SELEX method can be used to select specific regions and specific aptamers

motifs. Consensus motifs often located in stem-loop structures are thought to mediate binding specificity. The first SELEX experiment on single stranded RNA oligonucleotides was published by Tuerk and Gold, when investigating the bacteriophage T4 DNA polymerase (Tuerk and Gold, 1990). For RNA aptamers, another selection process has been facilitated and accelerated by Cox and co-workers, who established an automated selection procedure based on magnetic beads. This process has been adjusted and optimized by several others (Giordano et al., 1999; Katzin and Colli, 1983). An automated selection procedure for RNA and DNA aptamers without magnetic beads has also been reported. Due to its 2 -hydroxyl group, however, RNA is easily subjected to hydrolysis by RNases, which can diminish its applicability if it is not stabilized by chemical modifications. An alternative is the employment of Spiegelmers (L-RNAs), which represent the mirror images of the naturally occurring D-RNAs. Since the L-forms of nucleic acids do not occur in nature, there are no enzymes to degrade them. DNA aptamers, lacking the 2 -hydroxyl group, are also more stable than unmodified RNAs in biological samples. To select DNA aptamers specific for Vaccinia virus (VACV), aptamer selection can be performed in two steps. An initial chromatography was performed on proteins from virus-free cell culture supernatant to eliminate aptamers binding to cell debris or media components. Subsequently, a second chromatography was carried out on resin-bound heat-inactivated VACV particles. For direct recovery of highaffinity-binding aptamers, the resin of the affinity column was physically segmented into slices and the amount of retained binding aptamers was estimated by quantitative real-time PCR. High concentrations of aptamers not eluted during the extended washing procedure were identified on several affinity column segments. As an alternative to the SELEX process with 7 to more than 30 selection and amplification cycles requiring large amounts of the target molecule, a new selection

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process was established. Based on reports about selecting functional oligonucleotides and the potential of oligonucleotides as non-Watson-Crick-type binders to peptides and proteins, a new one-step aptamer isolation protocol called MonoLEX is used. This is used to retrieve DNA aptamers that have the potential to bind specifically to virus particles. The MonoLEX approach combined a single affinity chromatography step with subsequent physical segmentation of the affinity resin and one single final exponential amplification step of bound aptamers. A schematic representation of SELEX is given in Fig. 4.1. Specific aptamers were selected from a combinatory library of oligonucleotides characterized by two flanking primers of known sequence and an internal region of 20 random nucleotide positions (N20 DNA library). Under adequate chromatographic conditions, like flow laminarity, sufficient capacity and homogeneity of the resin, high-affinity-binding aptamers stuck to the target, whereas weakly binding oligonucleotides could be removed from the resin by ample washing. Since the selection is an in vitro process, depending on the final application of the aptamer, the selection conditions can be adapted accordingly. This procedure exploits the advantages of automation (i.e., it allows for a fast, reproducible, and parallelized selection of DNA aptamers). Only little manual handling is required. Merely the preparation of the solutions, precipitation, and amplification steps have to be performed manually. All other selection steps, like coupling of the target to the solid surface, interaction of DNA with the target molecule, wash and elution steps, as well as the single-stranded DNA preparation can be carried out automatically (Wochner et al., 2008). In literature we can now identify a large number of aptamers selected for a very broad spectrum of targets. Furthermore, the first aptamer has recently successfully reached the pharmaceutical market. This is an anti-VEGF aptamer for the treatment of macular degenerative disease that exerts its action by attacking the neo-vascularization characteristic of this disease, under the name of Macugen (Eyetech/Pfizer) (Ng et al., 2006; Kourlas and Schiller, 2006).

4.4 Applications of DNA and RNA Aptamers Aptamers offer the utility for biotechnological and therapeutic applications (Fig. 4.3) as they offer molecular recognition properties that reveal the commonly used biomolecule, antibodies. Aptamers are functional molecules, usually DNA or RNA oligonucleotides, with the appropriate sequence and structure to form a complex with a target molecule. They are able to bind tightly and selectively to disease markers and can greatly benefit disease diagnosis and therapy. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube. They are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. When compared to antibodies, aptamers have advantages and disadvantages as therapeutic and biological reagents (Jayasena, 1999). Aptamers present faster tissue penetration and wider applicability

4.4

Applications of DNA and RNA Aptamers

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Fig. 4.3 Aptamers have diverse applications in the field of diagnostics and therapeutics

and present the opportunity for simple base modifications to improve functionality by comparison. Furthermore their small size (molecular weights between 3,000 and 20,000) may reduce steric hindrance, increasing surface coverage during immobilization (Deng et al., 2001). The in vitro selection process (SELEX) can be more precisely monitored than can organismal immunization, and the affinities and specificities of aptamers can thus be better tailored than can those of antibodies. Aptamers can be more readily engineered than antibodies for biological/medicinal use (Jayasena, 1999). Their application is very diverse. Aptamers have been used as molecular recognition elements in analytical systems for detection, separation, or purification of target molecules. They also play an important role in medical therapy (Pan et al., 1995) and environmental analysis (Homann and Göringer, 2001). Aptamers have been selected for various purposes, making use of their high specificity, versatility and affinity in target recognition. Examples include aptamers that recognize proteins, peptides, dyes, amino acids, nucleotides, and drugs among others, to act as biosensors (Potyrailo et al., 1998), probes (Pavski and Le, 2001) and anti-clotting agents (Bock et al., 1992), whilst the first aptamer has made it to the market gaining FDA approval.

4.4.1 Aptamers for Detection of Pathogens Aptamers promise to be such additional candidates for prophylaxis and treatment of infectious diseases, as they can be directed against a wide variety of target molecules like toxins or even complete microorganisms (Xu and Ellington, 1996; Hamm,

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1996). Like the antibodies, aptamers bind to their target by their three-dimensional structure with high affinity and specificity. Compared to antibodies, aptamers have several advantages, as they are selected in vitro which also enables selection against toxic or weakly immunogenic targets. Especially in scenarios where infections with new, emerging pathogens have to be detected or treated, a fast technique providing specific detection tools may be helpful. The spectrum for different applications of aptamers like A38, either on a sensor for virus detection or for treatment of infections, requires an extremely high stability of the aptamer. The use of DNA aptamers facilitates such a high stability without modifications, even in body fluids. Many different aptamers have been selected by various SELEX-based protocols for a wide variety of targets ranging from small molecules to whole cells and bacteria. Some of these aptamers were shown to possess inhibitory activity with certain HIV strains (McEver, 1995), to block cell binding of human cytomegalovirus (CMV) (Bevilacqua et al., 1994) or influenza virus hemagglutinin (Hicke et al., 1996) or were selected as tools for differentiation of closely related influenza strains (Almeida-de-Faria et al., 1999). Some other examples include aptamers to HIV related targets (Andreola et al., 2001; de Soultrait et al., 2002; Duzgunes, 2001), hepatitis C (Biroccio et al., 2002; Hwang et al., 2000; Vo et al., 2003), Trypanosoma cruzi (Homann and Göringer, 1999; Homann and Göringer, 2001; Ulrich et al., 2002), prion proteins (Sayer et al., 2004; Weiss et al., 1997), thrombin (Bock et al., 1992; Griffin et al., 1993; Holland et al., 2000), anti-angiogenesis vascular endothelial growth factor (Blank et al., 2001; White et al., 2003), prostate specific membrane antigen (Lupold et al., 2002), Botulinum neurotoxin type A (Lou, 2009).

4.4.2 Aptamers for Therapy The most important challenge in fighting almost any disease is to be able to detect and treat it in the early stages. For most diseases there are changes in gene expression and subsequent protein products that could be used for detection. However, diseaseinitiated changes often occur in the depths of our tissues and are not visible from the surface. Therefore another challenge for the developing new technology to fight disease is to find ways of non-invasive imaging (e.g. no biopsy or surgery) of the body’s status and to find ways to target small sites inside the body where the disease process is initiating (Szpechcinski and Grzanka, 2006). Due to the high degree of specificity and targeted nature of aptamer behaviour towards the target molecule, it is expected that the dosage of therapeutic aptamer to be used can be the smaller, this strategy may reduce costs and alleviate bystander effects caused by the aptamer-conjugate toxic side effects (Gewirtz, 1999). Furthermore, more pharmokinetic data is readily available that demonstrates aptamer quick clearance. PEGylation is currently been studied to improve aptamer circulation for longer periods of time (Pendergrast et al., 2005). DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165) have been reported recently (Ikebukuro et al., 2007). These specific DNA aptamers against VEGF165 would be a useful sensing element

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Applications of DNA and RNA Aptamers

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for cancer diagnosis. These small single-stranded nucleic acids are claimed to act as mimics of antibodies in that they can recognise molecular targets with high specificity and are able to carry therapeutic agents, radioisotopes, directly to solid tumour masses or to individual cells that may have metastasised with greater efficiency (Brody and Gold, 2000; Cerchia et al., 2002; Sooter and Ellington, 2002). The antiVEGF aptamer pegaptanib has already received FDA approval for treatment for human ocular vascular disease (Ulrich et al., 2006). Another success story is the E2F decoy aptamer that binds the transcription factor E2F, aimed at reducing the proliferation of cardiac and vascular cells in cardiovascular diseases as well as in the development of malignancies (Fabbrizio et al., 1999). This aptamer is a naturally developed aptamer, because it contains a known consensus sequence that is found naturally in the human body. This aptamer is currently in Phase three trials study by Corgenetech, Inc. and being evaluated for its efficacy at restraining coronary and peripheral vascular graft failure. Aptamers also present the possibility to work as antidotes and more importantly the potential of creating a self-antidote, simply through the creation of a selfaptamer that disrupts the interaction with the target, thus allowing for a controlled therapy. This feature is not usually present in any of other biological therapy. However, drawbacks such as their cost, synthesis, bioavailability, pharmacokinetics properties and delivery hindered aptamers from immediate application in the pharmaceutical industry and more importantly the fact that antibodies are a proven biological therapeutic and thus aptamers would need to be more flexible, more stable and more efficient than antibodies in order to replace the later. Thus, aptamer development needs to focus on their advantages in relation to other more proven biological therapeutics and the possibilities they present.

4.4.3 Aptamers to Detect Change in Gene Expression Aptamers can also be used to detect a change in gene expression by binding the protein product of the gene. Aptamers are being developed in two ways to detect changes in gene expression in vivo. The first is to prepare probes in which the aptamer is part of a regulated nucleic acid sequence that is complementary to a particular mRNA. The second is to develop allosteric aptamers that will bind specific cell surface proteins that are the result of altered gene expression. Binding the cell surface protein brings about a structural change in the aptamer that result in its binding an imaging agent or drug to treat the disease. Many aptamers have been selected to recognize specific proteins. Some of these aptamers are currently in use clinically or are being investigated in clinical trials for treating disease. Allosteric aptamers called “cis-linked aptamers for medical procedures” (CLAMPs) are being developed that will recognize a particular cell surface protein and respond by binding another ligand once it has bound to the surface of a diseased cell. The other ligand could be a radiolabeled ligand that can be used to detect the diseased cell. Or it could be a prodrug that will release a toxic compound when brought near to the diseased cell.

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4.4.4 Aptamers as Diagnostics and Therapeutics The idea of using aptamers as therapeutic molecules have been suggested by several groups. Now aptamers are at the forefront of the list of the most promising agents as potential nucleic acid pharmaceuticals. A whole series of aptamers that can bind protein targets inside and outside the cell can be found in the literature, which illustrates the potential of DNA or RNA aptamers as therapeutic modalities. These aptamers are suitable for applications based on molecular recognition of a target molecule including diagnostics and therapeutics (Ulrich et al., 2006). The use of the SELEX method has been extended to complex targets such as red blood cell membranes (Morris et al., 1998) and the membrane-bound nicotinic acetylcholine receptor, whole virus particles (Pan et al., 1995) and African trypanosomes (Homann and Göringer, 1999). These ligands have dissociation constants in the picomolar to low nanomolar range for their protein targets. The SELEX methodology has also successfully been used to develop RNA antagonists of CD4 epitopes on mononuclear mouse cells (Davis et al., 1998) and selectin cell surface molecules (O’Connel et al., 1996; Jenison et al., 1998). These selected RNA antagonists prevented the P-selectin-dependent neutrophil-platelet adhesion in vitro. Now, RNA or DNA aptamers can be obtained for almost every target whether complex or small. The potential utility of aptamers as therapeutic agents is considerably enhanced by chemical modifications that lend resistance to nuclease attack (Davis et al., 1998; Ito et al., 1998). This resistance is either achieved by the addition of phosphorothioates (Andreola et al., 2001) or by the substitution of the 2 -OH groups of pyrimidines with 2 -F, 2 -NH2 , or 2 -OMe. Aptamers recognize epitopes with the same specificity as antibodies but in contrast to antibodies they possess low immunogenicity and are not subject to proteolytic degradation. The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and realtime PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner (Nitsche et al., 2007).

4.4.5 Aptamers as Radiopharmaceutical Tools The advent of techniques such as SELEX has allowed the study and manipulation of nucleic acids and has propelled the discovery of molecular tools for the treatment of human disease. Equipped with these new tools, the implementation of simple steps for the formation of a new class of therapeutic agents was one step away. The idea of attaching radionucleotides to a biological molecule is not new (Bernardo-Filho et al., 2005). Long and detailed studies have been carried out in the past with some success with antibodies, peptides or antisense targeting. Aptamer labelling was a natural step

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in the direction of molecular targeted radiopharmaceutical development, (Ferreira and Missailidis, 2007) propelled both by the understanding of the biological properties of these molecules, specially their chemical malleability and pharmacokinetics, and the simplicity of the chemistry involved in the labelling of aptamers for pharmacoimaging and pharmacotherapy (Britz-Cunningham and Adelstein, 2003; Cerchia et al., 2002; Urbain, 1999). Two DNA aptamers directed at two separate exosites on the human alphathrombin have been evaluated for their potential as thrombus imaging agents. Both aptamers were analysed and ODN2 was found to be suitable for imaging with rapid clearance rates. However, the conclusions of the study also found that the slow mass transfer to the clot worked against the in-vivo thrombin-dependent imaging (Doughan et al., 2003). More recently an aptamer that binds to the extracellular matrix protein Tenascin-C was 99mTc-labeled and analysed for the potential use as radiopharmaceutical in a variety of animal models. In this study rapid uptake and rapid clearance of the radiolabeled aptamer from blood and other tissues provided clear tumour imaging, thus suggesting potentially imaging and therapeutic applications (Hicke et al., 2006).

4.5 Conclusion When the first aptamers were developed 15 years ago, it was predicted that they had an enormous potential as a feasible alternative to antibodies. Although the application of antibodies for the detection and therapy of infectious agents is well established, aptamers have some potential advantages over antibodies that may fill the gaps that antibody-based applications possess and that expedite the use of aptamers in the virologist’s laboratory. These advantages include their small size, eased cell penetration, rapid and cheap synthesis including a variety of chemical modifications and the fact that aptamers are non-immunogenic. Probably their crucial benefit is a selection process performed in vitro. Thus, aptamers can be selected against targets that are either weakly immunogenic or toxic, like toxin proteins. These targets are usually not applicable for in vivo production of monoclonal antibodies in mice; however, high affinity aptamers have been selected against some of these problematic targets. Moreover, the chemical and physical conditions for aptamer selection can be adapted to the real environment in which the aptamer will finally be applied. This includes cross-selection against similar targets that have to be excluded from an aptamer’s detection pattern. With the FDA approval of the first aptamer-based drug Macugen and the recent publication of an aptamer-based cocaine sensor, aptamers are starting to tap their full potential with the increasing need for additional specific detection tools. With the improving automation of the aptamer selection process and the growing knowledge of pathogen-specific targets, the number of aptamers used in diagnostics and therapy of infectious diseases will dramatically increase in the future.

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Chapter 5

Reporter Gene

Abstract Reporter genes have become an invaluable tool in studies of gene expression. Reporter gene technology is widely used to monitor the cellular events associated with signal transduction and gene expression. Based upon the splicing of transcriptional control elements to a variety of reporter genes (with easily measurable phenotypes), it “reports” the effects of a cascade of signaling events on gene expression inside cells. The principal advantage of these assays is their high sensitivity, reliability, convenience, and adaptability to large-scale measurements. They are widely used in biomedical and pharmaceutical research and also in molecular biology and biochemistry. Keywords Reporter · Gene · Signaling · Inducer · Therapeutics · Luc · X-gal · GFP · Lac · Ruc · HGH · CAT · GUS · LacZ · Promoter assay · Promoter · Molecular imaging · Reporter gene construct · Reporter gene assay

5.1 Prologue Developments in molecular genetics have given us insights, at the molecular level, into vital processes in living organisms, such as embryonic development, growth regulation, differentiation, pathogenesis, and carcinogenesis. Insights into the mechanism of pathologic processes, such as developmental disorders and carcinogenesis, have stimulated efforts to develop therapeutic approaches to prevent or correct these processes. Techniques to directly change the genetic information of a cell have raised high expectations of the therapeutic potential of genetic manipulation. In vitro, genes have been introduced successfully into cells, thus changing the genotype and the phenotype of cells. These developments have raised hopes that diseases appearing to be incurable can soon be cured.

5.2 Concept Genetic reporter systems represent an extensive toolbox for the study of regulatory promoter and enhancer sequences as well as for the study of transcription factors M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_5,

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(Alam and Cook, 1990; Kain and Ganguly, 2001). A gene consists of two functional parts, one is a DNA-sequence that gives the information about the protein that is produced (coding region). The other part is a specific DNA-sequence linked to the coding region; it regulates the transcription of the gene (promoter). The promoter is either activating or suppressing the expression of the gene. A reporter gene is one that codes for a product that is easy to measure. These genes are often used in situations in which the product of some gene of interest is difficult to assay quantitatively. They are used to replace other coding regions whose protein products are difficult to assay. The most typical use of a reporter gene is to analyze how a gene is regulated. Let’s consider a typical gene as being composed of a regulatory region, lying upstream of the transcription start point, and a structural region, including the open reading frame and any 5 or 3 untranslated regions (UTR’s). Fig. 5.1 The reporter gene affects the expression of the structural gene

The reporter gene would typically be attached to the regulatory region, using recombinant DNA methods, making an operon fusion like the second one shown in Fig. 5.1. In this arrangement, anything that ordinarily affects the expression of the natural gene would also affect the expression of the reporter gene. Among the more commonly used reporter genes are shown in Table 5.1. The purpose of the reporter gene assay is to measure the regulatory potential of an unknown DNA-sequence. This can be done by linking a promoter sequence to an easily detectable reporter gene such as that encoding for the firefly luciferase. Reporter genes have become an invaluable tool in studies of gene expression (Naylor, 1999). They are widely used in biomedical and pharmaceutical research and also in molecular biology and biochemistry. A reporter gene enables researchers to track and study another gene in cell cultures, animals, and plants. Because most gene therapy techniques only work on a small number of individuals, researchers need to use a reporter gene to identify which cells have taken up the gene currently under study, and which have incorporated it into their chromosomes. Regulatory sequences of interest are combined with a reporter construct of choice and are subsequently assayed in conjunction with relevant transcription factors (Phippard and Manning, 2003). Reporter genes can be attached to other sequences so that only the

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reporter protein is made or so that the reporter protein is fused to another protein (fusion protein). Reporter genes can “report” many different properties and events: ◦ the strength of promoters, whether native or modified for reverse genetics studies; ◦ the efficiency of gene delivery systems; ◦ the intracellular fate of a gene product, a result of protein traffic; ◦ interaction of two proteins in the two-hybrid system or of a protein and a nucleic acid in the one-hybrid system; ◦ the efficiency of translation initiation signals; ◦ and the success of molecular cloning efforts.

Table 5.1 Activity of some reporter genes Reporter genes and protein

Activity and measurement

GUS (β-glucuronidase)

Transfers radioactive acetyl groups to chloramphenicol; detection by thin layer chromatography and autoradiography Hydrolyzes colorless galactosides to yield colored products

CAT (chloramphenicol acetyltransferase) GAL (β-glucuronide) LUC (luciferase) GFP (green fluorescent protein)

Hydrolyzes colorless glucuronides to yield colored products Oxidizes luciferin, emitting photons Fluoresces on irradiation with UV

If the reporter system is well chosen then the level of reporter gene expression will correlate with the transcriptional activity of the introduced transgenic factors. In order to assure such a correlation, it is important that the reporter gene does not disturb the metabolism of the transformed cells and that the gene is not endogenously expressed by the target cells creating background signals. Expression of reporter genes can be measured by enzyme activity assay of the expressed enzyme encoded by the reporter gene using chromo-, fluoro- or luminogenic substrates; immunological assay of the expressed protein encoded by the reporter gene (reporter gene ELISA); or by histochemical staining of cells or tissues typically to localize enzymatic activity ectopically expressed from reporter gene constructs in transformed cells. Reporter genes are widely used to study gene expression and regulation mechanisms in living cells. Not all expressed enzymes are easily detectable, so reporter genes were introduced into cellular DNA to investigate gene function by means of a measurable property, the luminescence. These reporter genes are for example: firefly luciferase, beta-glycosidase, alkaline phosphate, beta-glucuronidase, betaglucosidase. By far the most popular reporter is the firefly luciferase from the American firefly (Photinus pyralis). The high sensitivity, easy handling, short process time and a high quantum yield of the bioluminescence reaction make this

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method to the “method of choice” to understand gene regulation. Typical commercial assay kits are optimized for extended half life time of more than five minutes. Most reporter genes are placed downstream genetically of the promoter region, but close to the gene under study. This ensures that these genes are expressed together, and are not separated during cell division by crossover events. Sometimes, reporter genes are simply placed in a vector independent of a chromosome, and other techniques used to identify the gene under study (Pardy, 1994). Reporter genes offer a big advantage, because one doesn’t need a separate assay for each regulatory region being studied. Also, for many reporter genes simple indicator plate assays have been developed that allow one to determine levels of expression by the colour of a bacterial or yeast colony, and perhaps to isolate mutants based on changes in the colour. For instance, a bacterial strain making substantial amounts of beta-galactosidase will form a blue colony on a plate with X-gal; this allows one to identify cells with different levels of beta-galactosidase, and to use X-gal plates for any regulatory region driving expression of beta-galactosidase.

5.3 Reporter Gene Assay Study of gene expression can be facilitated by using a reporter gene assay (Fig. 5.2). Instead of directly measuring the level of target gene mRNA, one can clone the promoter region of the gene of interest in front of a reporter gene and measure the reporter gene expression as a reflection of the expression of the gene of interest (Kain and Ganguly, 2001). A simple lacZ-fusion system is to measure the activity of the reporter gene product β-galactosidase (Thibodeau et al., 2004). Different strategies of making the fusion construct and their applications have been reported. This method is particularly useful to dissect the promoter region of the gene of interest and is also used in other experimental protocols such as the yeast two-hybrid analysis. To introduce a reporter gene into an organism, scientists place the reporter gene and the gene of interest in the same DNA construct to be inserted into the cell or organism. For bacteria or eukaryotic cells in culture, this is usually in the form of a circular DNA molecule called a plasmid. It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest. Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent proteins; examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under UV light, and the enzyme luciferase (Phippard and Manning, 2003; Nordeen, 1988), which catalyzes a reaction with a luciferin to produce light. Another common reporter in bacteria is the lacZ gene, which encodes the protein β-galactosidase. This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal (an inducer molecule such as

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Fig. 5.2 Reporter gene can be used as a molecular diagnostic tool

IPTG is also needed under the native promoter). An example of a selectablemarker reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene (Altschmied and Duschl, 1997; Pardy, 1994), which confers resistance to the antibiotic chloramphenicol. Various detection methods (Jeffereson, 1989; Gillissen et al., 1998; Nordeen, 1988; Altschmied and Duschl, 1997) are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence.

5.3.1 Luc Reporter Gene System In nature, the luciferase reaction occurs in the peroxisomes of a specialized light organ in fireflies (Photinus pyralis). The luciferase reaction emits a yellow-green light (560 nm) and requires the co-factors ATP, Mg2+ , O2 and the substrate luciferin. The glow is widely used as an assay for luciferase activity to monitor regulatory elements that control its expression (Olesen et al., 2002). Luc is particularly useful as a reporter gene since it can be introduced into living cells and into whole organisms such as plants, insects, and even mammals. Luc expression does not adversely affect the metabolism of transgenic cells or organisms. In addition, the luc substrate luciferin is not toxic to mammalian cells, but it is water-soluble and readily transported into cells. Since luc is not naturally present in target cells the assay is virtually background-free. Hence, the luc reporter gene is ideal for detecting low-level gene expression (Nordeen, 1988). A second reporter system based on luciferase expressed by the ruc gene from Renilla (Renilla reniformis) has also become available (Wang et al., 2002). The activities of firefly and Renilla luciferase can be combined into a dual reporter gene assay.

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Luciferase expression is measured by adding ATP and luciferin to cell lysates and then analyzing bioluminescence by spectroscopy or with a scintillation counter allowing for the detection of even a few hundred thousand enzyme molecules. The development of camera based imaging systems to visualize luc gene expression in vivo has made rapid progress; most impressively, in the generation of transgenic mice carrying the luc gene. In some of these mice strains luc expression is triggered by intoxication or the inflammation processes. The effects of disease to mice as well as the results of therapy can be studied non-invasively and in real time (www.xenogen.com). Luciferin was initially isolated by extraction from Photinus pyralis which is a tedious process. Despite manufacturing bulk quantities of luciferin chemically, commercial grade luciferin is still specified “synthetic”.

5.3.2 Ruc Reporter Gene System Renilla luciferase serves as a marker protein in bioluminescent fusion constructs of the ruc gene (Wang et al., 2002). It is similar to the above described luc vectors which is useful in low-light imaging of gene expression and of regulation processes in living cells as well as in transgenic plants. Coelenterazine is the best substrate for Renilla luciferase generating 40% more light than native coelenterazine with a considerably higher initial light intensity (Yu and Szalay, 2002). The firefly luciferase gene (luc) has proven to be very useful in reporter gene research and drug discovery assays, when used with luciferin and ATP: firefly luciferase/luciferin emits light at 560 nm, Renilla luciferase/coelenterazine emits light at 475 nm. Because these two reporter systems emit light at quite different wave lengths, it is possible to use fire fly luciferase/luciferin and Renilla luciferase/coelenterazine as a dual reporter system. In dual reporter gene systems two distinct reporter enzymes can be measured within one sample, whereby the second reporter is often used as an internal control. Dual reporter luminescence assays preferably using firefly luciferase as the first reporter enzyme, have become very popular, e.g. for measuring transcriptional activity in studies of the structural or physiological basis of regulated gene expression (Yu and Szalay, 2002).

5.3.3 Green Fluorescent Protein (GFP) Reporter Gene System The green fluorescent protein from the Aequorea victoria jellyfish emits green fluoresence without the need for any enzyme or co-factors (Phillip, 2001). Gene expression and protein localization is possible in situ and in vivo: no secondary transformation is required. Reporter gene expression can be qualitatively monitored live e.g. for the study of dynamic processes inside the cell (Chalfie et al., 1994). There are a number of GFP proteins in use. Selecting GFP with minimal

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overlap in light emission allows dual reporter gene studies, for instance, to localize different sites of expression or, the relative effects of different regulatory proteins (Chalfie, 1995). Most recently, bioluminescence resonance energy transfer (BRET) detection systems have been introduced. These systems are based on excitation energy transfer from a bioluminescent donor molecule, e.g. Renilla luciferase to a fluorescent acceptor molecule, like green fluorescent protein (GFP). It is known that luciferase interacts rapidly with GFP in a highly specific manner to form an equilibrium complex responsible for this very efficient energy-transfer phenomenon. Renilla luciferase as well as GFP are both attached to the proteins to be examined (Yu and Szalay, 2002), e.g. by chemical synthesis or by use of genetic engineering methods. In the latter case the gene fusion constructs are used to transfect or transform cells and express the fusion proteins. In the presence of coelenterazine, a BRET signal is generated when the fusion proteins are associated. Excellent discrimination of the ruc and GFP signals results when coelenterazine is used as the ruc substrate (Wang et al., 2002). As the efficiency of the non-radioactive energy transfer from the Renilla luciferase fusion construct to the GFP conjugate depends on steric effects, for instance, on the distance of the protein molecules and on their relative orientation, the method can be used to study protein–protein interaction as well as to screen for inhibition of such an interaction: in drug discovery applications or receptor-ligand studies. The new spectroscopic techniques and assay methods built on BRET are fascinating tools for investigation in vivo.

5.3.4 Human Growth Hormone (hGH) Reporter Gene System The human growth hormone (hGH) encoded reporter protein is secreted into the culture medium by transfected cells (Selden et al., 1986). Thus no cell lysis is required for detection. Detection of the secreted hGH requires the use of hGH antibodies. Such antibodies used to be radiolabeled in order to allow detection of the antibody hGH complex. Today hGH is assayed by indirect ELISA. First, the hGH from the supernatant of the culture medium binds to the antibody on the plate. Subsequently, the bound hGH is detected in two steps via a digoxigenincoupled anti-hGH antibody and a peroxidase-coupled anti-digoxigenin antibody. Bound peroxidase is quantified by incubation with a peroxidase substrate such as TMB (3,3 ,5,5 -tetramethylbenzidine,T-2100; Selden et al., 1986).

5.3.5 Beta-Galactosidase Reporter Gene System β-galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins. Alternate or nicknames

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are “beta-gal” or “β-gal”. Lactase is often confused as an alternate name for β-galactosidase, but it is actually simply a sub-class of β-galactosidase (Thibodeau et al., 2004). The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via “shallow” and “deep” binding. Monovalent potassium ions (K+ ) as well as divalent magnesium ions (Mg2+ ) are required for the enzyme’s optimal activity. The beta-linkage of the substrate is cleaved by a terminal carboxyl group on the side chain of a glutamic acid. In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction. However, it is now known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl intermediate. In humans, the nucleophile of the hydrolysis reaction is Glu-268 (McCarter et al., 1997). β-galactosidase is measured by a photometric assay (Pardy, 1994).

5.3.6 CAT Reporter Gene System Microbial chloramphenicol acetyltransferase (CAT) catalyzes the transfer of acetyl groups from acetyl coenzyme A to chloramphenicol and represents the oldest reporter gene system still in broad use today. The assay is performed by incubating transformed cells with chloramphenicol carrying a radioactive label. CAT activity can be assayed by autoradiography of lysate subjected to thin layer chromatography (TLC). Acetylated and non-acetylated chloramphenicol can readily be separated by TLC of lysate. Hence, the presence of labeled acetylated chloramphenicol correlates to the expression of CAT (Zhang et al., 2003). In an effort to limit the use of radioactivity in the lab the method is rapidly being replaced by immunological detection of the CAT enzyme by ELISA.

5.3.7 LacZ Reporter Gene System Reporter genes have become standard genetic tools used to evaluate either the transcriptional or the translational activity associated with genes of interest, whose products cannot be easily assayed. The lacZ gene from E. coli has been used very effectively to quantify such regulated activities in many different organisms. Colonies expressing lacZ appear blue on culture media containing X-gal (B-7150). The prokaryotic beta-galactosidase encoded by lacZ from the lac operon catalyzes the hydrolysis of beta-galactosides, most prominently the hydrolysis of lactose. Galactosidase activity in lysates of transfected cells can be assayed via absorption of o-nitrophenolate from o-nitrophenyl-ß-D-galactoside (ONPG,N-4190), fluorescence of 4-methylumbelliferone (from 4-methyl-umbelliferyl-ß-galactopyranoside, M-5550), or via chemiluminescence of 1,2-dioxetan-galactopyranoside derivatives. The lacZ is often co-transfected together with other reporter constructs such as luciferase serving as an internal control.

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The lacZ reporter gene is widely used as a convenient tool to identify recombined plasmids during molecular cloning procedures. Since the complete lacZ gene is too large to be carried by a plasmid, cloning vectors contain only the alpha region of the lacZ gene. Expression of the alpha region of lacZ yields an inactive protein. However, the truncated protein encoded by the alpha region can combine into active beta-gal when complemented with a protein from lacZ lacking the alpha region. Such “intragenic complementation” can be achieved if a plasmid carrying the alpha region of lacZ is inserted into a bacterial cell containing a mutated alpha region of the lacZ gene. These cells will turn beta-gal positive if the plasmid is present. DNA cloning into lacZ vectors occurs such that the fragment is likely to insert into the alpha lacZ sequence on the plasmid which renders the alpha-region dysfunctional. In this case intragenic complementation no longer occurs. Hence, cells carrying plasmids containing foreign DNA will remain beta-gal negative. Beta-gal negative cells can readily be isolated from positive ones by incubation of bacteria on chromogenic culture media containing X-gal (5-bromo-4-chloro-3-indoxyl-betaD-galactopyranoside, B-7150) and IPTG (isopropyl-beta-D-thiogalactopyranoside, I-8000) followed by manual selection of the colorless colonies.

5.3.8 GUS Reporter Gene System GUS encodes the beta-glucuronidase enzyme from E. coli and it has been extensively used as a marker gene (Jefferson, 1989). The main advantage of the GUS reporter gene system is the stable expression of GUS enzyme in plant cells (Gilissen et al., 1998). GUS activity can readily be detected by chromogenic or fluorogenic enzyme substrates. Typically, very little background signal is encountered because the intrinsic expression of endogenous beta-glucuronidase genes is not significant in plants. In addition, plant metabolism remains largely unaffected by the presence of E. coli beta-glucuronidase. Various beta-glucuronic acid substrates are available for detection of GUS expression. The most widely used fluorogenic substrate for detection of beta-glucuronidase activity in vitro is 4-methylumbelliferyl beta-D-glucuronide (MUG, 4-methylumbelliferyl-beta-D-glucuronic acid dihydrate, M-5700). For other applications the use of X beta-glucuronide (5-bromo4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt, B-7300), which yields an insoluble blue precipitate in the presence of beta-glucuronidase, has become standard.

5.4 Transformation and Transfection Assays Transfection and transformation are the basic two ways of expressing a foreign or modified gene in an organism. They are effective in only a small percentage of a population subjected to the techniques. Thus, a method for identifying those few successful gene uptake events is necessary. Reporter genes used in this way are

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normally expressed under their own promoter independent from that of the introduced gene of interest; the reporter gene can be expressed constitutively (that is, it is “always on”) or inducibly with an external intervention such as the introduction of IPTG in the β-galactosidase system. As a result, the reporter gene’s expression is independent of the gene of interest’s expression, which is an advantage when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access. In the case of selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol. Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions (Zhang et al., 2003).

5.5 Gene Expression Assays Reporter genes can also be used to assay for the expression of the gene of interest, which may produce a protein that has little obvious or immediate effect on the cell culture or organism. In these cases the reporter is directly attached to the gene of interest to create a gene fusion. The two genes are under the same promoter and are transcribed into a single messenger RNA molecule. The mRNA is then translated into protein. In these cases it is important that both proteins be able to properly fold into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.

5.6 Promoter Assays Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism. In this case there is no separate “gene of interest”; the reporter gene is simply placed under the control of the target promoter and the reporter gene product’s activity is quantitatively measured. The results are normally reported relative to the activity under a “consensus” promoter known to induce strong gene expression (Koken et al., 1994). The use of reporter enzyme provides a rapid and sensitive method for the analysis of transgene expression.

5.7 Role of Reporter Gene Assay in Molecular Diagnostics Reporter gene assays have emerged as a very general strategy for indirectly monitoring various intracellular events. Furthermore, reporter genes are being used to monitor gene/cell therapies, including the location(s), time variation, and magnitude of gene expression. The reporter gene assay has been used for the detection of

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pathogens (Collins and Franzblau, 1997). Rubella virus was detected using replicon based reporter gene assay (Tzeng et al., 2005). Influenza A virus replication was also detected by virus inducible reporter gene (Lutz et al., 2005). Some progress was reported in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods (Deo and Daunert, 2001). Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission (Shaner et al., 2005). Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein Inouye and Tsuji, 1994. The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening (Tsien, 1998). However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds (Changsen et al., 2003). In another study a throughput screening of G protein coupled receptors were performed using reporter gene assay (Durocher et al., 2000). Most current molecular genetic imaging strategies are “indirect,” coupling a “reporter gene” with a complimentary “reporter probe.” Reporter gene constructs can be driven by constitutive promoter elements and used to monitor gene therapy vectors and the efficacy of transgene targeting and transduction, as well as to monitor adoptive cell-based therapies. Inducible promoters can be used as “sensors” to regulate the magnitude of reporter gene expression and can be used to provide information about endogenous cell processes. Reporter systems can also be constructed to monitor mRNA stabilization and specific protein-protein interactions. Promoters can be cell specific and restrict transgene expression to certain tissue and organs (Serganova and Blasberg, 2005). Molecular imaging is a relatively new discipline, which developed over the past decade, initially driven by in situ reporter imaging technology (Blasberg, 2003). Human reporter genes will play an increasingly more important role in the clinical application of positron-emission-tomography-based reporter gene imaging

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(Serganova et al., 2007). Examples of highly expressed cell membrane receptors include specific membrane somatostatin receptors (hSSTrs). The transporter group includes the sodium iodide symporter (hNIS) and the norepinephrine transporter (hNET). The endogenous enzyme classification includes human mitochondrial thymidine kinase 2 (hTK2). Initial applications of reporter gene imaging in patients deal with gene therapy and adoptive cell-based therapies. These studies from the availability of efficient human reporter systems can provide critical monitoring information for adenoviral-based, retroviral-based and lenteviral-based gene therapies, oncolytic bacterial and viral therapies, and adoptive cell-based therapies. Translational applications of noninvasive in vivo reporter gene imaging are likely to include: (a) quantitative monitoring of gene therapy vectors for targeting and transduction efficacy in clinical protocols by imaging the location, extent and duration of transgene expression; (b) monitoring of cell trafficking, targeting, replication and activation in adoptive T-cell and stem/progenitor cell therapies; (c) and assessments of endogenous molecular events using different inducible reporter gene imaging systems.

5.8 Conclusion Reporter gene technology is widely used to monitor the cellular events associated with signal transduction and gene expression. Based upon the splicing of transcriptional control elements to a variety of reporter genes (with easily measurable phenotypes), it “reports” the effects of a cascade of signalling events on gene expression inside cells. The principal advantage of these assays is their high sensitivity, reliability, convenience, and adaptability to large-scale measurements. With the advances in this technology and in detection methods, it is likely that luciferase and green fluorescent protein will become increasingly popular for the non-invasive monitoring of gene expression in living tissues and cells. Such techniques will be important in defining the molecular events associated with gene transcription, which has implications for our understanding of the molecular basis of disease and will influence our approach to gene therapy and drug development. The future appears to be promising for the continued expansion of the use of reporter genes in the many evolving biomedically related arenas (Min and Gambhir, 2008).

References Alam, J., Cook, J.L., 1990, Reporter genes: application to the study of mammalian gene transcription. Anal Biochem 188:245–254. Altschmied, J., Duschl, J., 1997, Set of optimized luciferase reporter gene plasmids compatible with widely used CAT vectors. Biotechniques 23:436–438. Blasberg, R.G., 2003, Molecular imaging and cancer. Mol Cancer Ther 2:335–343. Chalfie, M., 1995, Green fluorescent protein. Photochem Photobiol 62:651–656. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C., 1994, Green fluorescent protein as a marker for gene expression. Science 263:802–805.

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Changsen, C., Franzblau, S.G., Palittapongarnpim, P., 2003, Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob Agents Chemother 47:3682. Collins, L., Franzblau, S.G., 1997, Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41:1004–1009. Deo, S.K., Daunert, S., 2001, Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis. Fresen J Anal Chem 369:258–266. Durocher, Y., Perret, S., Thibaudeau, E., Gaumond, M.H., Kamen, A., Stocco, R., Abramovitz, M., 2000, A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture. Anal Biochem 284:316–326. Gilissen, L.J., Metz, P.L., Stiekema, W.J., Nap, J.P., 1998, Biosafety of E. coli beta-glucuronidase (GUS) in plants. Transgenic Res 7:157–163. Inouye, S., Tsuji, F., 1994, Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein. FEBS Lett 341:277–280. Jefferson, R.A., 1989, The GUS reporter gene system. Nature 342:837–838. Kain, S.R., Ganguly, S., 2001, Overview of genetic reporter systems. Curr Protoc Mol Biol Chapter 9:Unit9.6. Koken, S.E., Greijer, A.E., Verhoef, K., van Wamel, J., Bukrinskaya, A.G., Berkhout B., 1994, Intracellular analysis of in vitro modified HIV Tat protein. J Biol Chem 269:8366–8375. Lutz, A., Dyall, J., Olivo, P.D., Pekosz, A., 2005, Virus-inducible reporter genes as a tool for detecting and quantifying influenza A virus replication. Virol Methods 126:13–20. McCarter, J., Burgoyne, D., Miao, S., Zhang, S., Callahan, J., Withers, S., 1997, Identification of Glu-268 as the catalytic nucleophile of human lysosomal beta-galactosidase precursor by mass spectrometry. J Biol Chem 272:396–400, PMID 8995274. Min, J.J., Gambhir, S.S., 2008, Molecular imaging of PET reporter gene expression. Handb Exp Pharmacol 185(Pt 2):277–303. Naylor, L.H., 1999, Reporter gene technology: the future looks bright. Biochem Pharmacol 58(5):749–757. Nordeen, S.K., 1988, Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6:454–458. Olesen, C.E., Yan, Y.X., Liu, B., Martin, D., D’Eon, B., Judware, R., Martin, C., Voyta, J.C., Bronstein, I., 2000, Novel methods for chemiluminescent detection of reporter enzymes. Methods Enzymol 326:175–202. Pardy, K., 1994, Reporter enzymes for the study of promoter activity. Mol Biotechnol 2: 23–27. Phillips, G., 2001, Green fluorescent protein–a bright idea for the study of bacterial protein localization. FEMS Microbiol Lett 204 (1):9–18. PMID 11682170. Phippard, D., Manning, A.M., 2003, Screening for inhibitors of transcription factors using luciferase reporter gene expression in transfected cells. Methods Mol Biol 225:19–23. Selden, R.F., Howie, K.B, Rowe, M.E., Goodman, H.M., Moore, D.D., 1986, Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol 6:3173–3179. Serganova, I., Blasberg, R., 2005, Reporter gene imaging: potential impact on therapy. Nuc Med Biol 32:763–780. Serganova, I., Ponomarev, V., Blasberg, R., 2007, Human reporter genes: potential uses in clinical studies. Nuc Med Biol 34:791–807. Shaner, N., Steinbach, P., Tsien, R., 2005, A guide to choosing fluorescent proteins. Nat Methods 2:905–909. Thibodeau, S.A., Fang, R., Joung, J.K., 2004, High-throughput beta-galactosidase assay for bacterial cell-based reporter systems. Biotechniques 36:410–415. Tsien, R., 1998, The green fluorescent protein. Annu Rev Biochem 67:509–44. PMID 9759496.

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Tzeng, W.-P., Zhou, Y., Icenogle, J., Frey, T.K., 2005, Novel replicon-based reporter gene assay for detection of rubella virus in clinical specimens. J Clin Microbiol 43:879–885. Wang, Y., Yu, Y.A., Shabahang, S., Wang, G., Szalay, A.A., 2002, Renilla luciferase-Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals. Mol Genet Genomics 268:160–168. Yu, Y.A., Szalay, A.A., 2002, A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo. Mol Genet Genomics 268:169–178. Zhang, K., Kurachi, S., Kurachi, K., 2003, Limitation in use of heterologous reporter genes for gene promoter analysis: silencer activity associated with the chloramphenicol acetyltransferase reporter gene. J Biol Chem, 278:4826–4830.

Chapter 6

Impact of HGP on Molecular Diagnostics

Abstract The Human Genome Project has heralded a whole new era in our understanding of the molecular basis of disease. New opportunities now arise to predict disease by genetic testing, and in some cases to prevent disease through surveillance or other specific interventions. Increasingly it will be possible to test for predisposition to disease, to develop new treatments or to tailor available treatments more specifically to an individual’s genetic make-up. Keywords Single gene disease · Human genome project · HGP · HUGO · Functional genomics · Individual drug treatment · Pharmacogenetics · Drug design · Systems biology · Genetic test · HapMap · Genome medicine

6.1 Prologue In the past decade, with the availability of technology and knowledge fuelled by the investment and interest in the Human Genome Project, molecular diagnostics has recently enabled laboratories to offer diagnostic and predictive tests for inherited disorders (Ferrari et al., 2003). Large amounts of genetic information can be determined at increasing efficiencies. The Human Genome Project (HGP) will change medicine and medical research irrevocably. The obvious gains in genetic knowledge from the HGP, together with the advances which will flow into bioinformatics, biotechnology and the potential for novel therapeutic agents, will ensure that the financial investment in the HGP is repaid many times over. The HGP’s costs in terms of ethical and social issues remain to be determined, but it is to be hoped that these will not detract from the scientific and medical achievements.

6.2 Origin of the Human Genome Project The Human Genome Project, the mapping of our 30,000–50,000 genes and the sequencing of all our DNA, will have major impact on biomedical research and the whole of therapeutic and preventive health care. The tracing of genetic diseases M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_6,

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to their molecular causes is rapidly expanding diagnostic and preventive options. The increased insights into molecular pathways, gained from high-throughput “functional genomics”, using DNA-chip and protein-chip approaches and specially designed animal model systems, will open great prospects for pharmacological and genetic therapies. Powerful bioinformatics and biostatistics will further improve our pattern recognition and accelerate progress. A rapidly expanding area of high expectations is that of “pharmacogenomics”: the design of more effective drugs with lower toxicity through tailoring of drug treatment to individual, genetically determined differences in drug metabolism. Not only will this decrease the cost of health care through reduction of adverse drug reactions, but a better stratification of populations will also provide more statistical power farther upstream in drug trials. However, the optimal benefits from the current explosion of “data mining” will only be realized when the basic data are made and kept publicly accessible, while at the same time safeguarding the protection of intellectual property arising from downstream inventions. This is one of the goals of HUGO, the international Human Genome Organization, established 13 years ago to assist coordination of data acquisition and exchange and societal implementation of the genome project. Additional points of attention in this historic endeavour are the prevention of stigmatization and discrimination and the safeguarding of a worldwide balance in the contribution by and benefits to different populations, while respecting the diversity in cultures and traditions (van Ommen, 2002). The Human Genome Project (HGP) is an effort to understand the genetic instructions that make up a human. The human genome is the DNA that resides within every human cell. By sequencing the genome, scientists will identify the location and composition of all of our 100,000 or so genes. The genes carry information for making the proteins that direct the make up the each human. Among other things, proteins control human development, physiology, and resistance to disease. Knowledge of gene sequences is tremendously beneficial to scientists studying all facets of living things. The complete genome sequence of human and many other species provides a new starting point for understanding our basic genetic make-up and how variations in genetic instructions result in human disease or other variants of human phenotypes (Peltonen, 2001). Realization of this goal will include characterization of all the genes that cause or predispose to disease, which will most certainly lead to the development of powerful new tools for diagnosis, prevention, and treatment in all medical fields.

6.3 Goals and Issues of HGP The HGP was conducted as collaboration between large-scale sequencing centres coordinated by the National Institute for Human Genome Research and the Department of Energy. Following the developmental phase resulting in genetic and physical maps and technology for high throughput sequencing, the US project

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was organized into 9 large-scale genomic sequencing centres distributed among universities and research institutes. As new technologies for understanding and working with DNA were developed in the 1980s, the idea arose to sequence the entire human genome systematically, and with this idea arose the HGP. HGP is an attempt to map completely the entire spectrum of genetic materials that can be found in all human beings. It is a research effort initiated by the DOE and jointly managed by the DOE and the National Institutes of Health (NIH). A unifying fact of human genetics is that all humans have genomes – the complete set of our genetic instructions on the 23 pairs of chromosomes within each of us – that are 99.9% identical in sequence. The DNA molecules that carry these genetic instructions are linear, information-containing molecules made up of four simple bases or building blocks – adenine, cytosine, guanine and thymine – that pair up to form the well-known DNA double helix. Although each one of our cells contains about six billion base pairs of DNA, three billion from each parent, we think that our differences are determined by only about one base pair in each thousand (Collins and Patrinos, 1998). The genome project now embraces three main technical goals: genetic linkage maps to trace the inheritance of chromosome regions through pedigrees; physical maps of large chromosome regions, to enable the direct study of DNA structure in search of genes; and substantial DNA sequence information, enabling the correlation of DNA changes with alterations in biological function. If history were logical, then the genome project would have grown from a discussion of each in turn and how to bring them together into a coherent plan. History is not logical, however, and it was DNA sequencing technology rather than genetic linkage mapping that gave rise to the idea of a human genome project. One goal of the HGP is to localize all of the estimated 80,000 genes on the human chromosomes and to determine the sequence of all three billion units of DNA that constitute one set of those chromosomes. This information will vastly accelerate studies that will characterize what those genes do and how disease can result from errors in their functioning. An important element of the HGP is to enable technologies that will allow biologists to uncover gene function more efficiently. This is important because while the HGP describe the human genome in molecular detail, its longer term and more profound impact will be to reveal critical mechanisms of human biology and supply the medical context within which investigations on the molecular pathology of human diseases can most efficiently take place. This will lead to a future medicine in which prevention will be firmly rooted in mechanistic knowledge and potential interventions can be more targeted and effective. The promise of the HGP (Fig. 6.1) goes far beyond medicine to many other areas of science. In addition to its many implications for medicine and human health which includes mutation detection, more accurate risk assessment, more precise disease diagnosis, more rapid characterization of genetic damage and repair processes, and the identification of precision pharmaceuticals based on intimate biological knowledge this international project is creating technologies and resources that will be applied to the characterization of the genomes of other living

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Fig. 6.1 Implication of HGP is to reach the mankind for better health and cheaper diagnostic treatment

organisms. This information will, in turn, provide us with important new practical applications in energy, environmental protection, agriculture, and industrial processes.

6.4 The Human Genome and Genetic Variation The genome contains the genetic information that is present in every cell in the body. The most important sequences in the genome (though only a small percentage of the total) are those that code for the production of proteins: the structural and functional units of all living cells. It is these sequences that correspond to the genes identified by classical genetics. While we all carry the same set of genes, their precise sequences may vary from individual to individual (that is, we carry different alleles), leading to variation in the structure or amounts of the associated proteins and thereby to variation in characteristics such as the colour of the hair or eyes (Risch et al., 2003). Of more importance when considering health and health care, these variations may also lead to differences in susceptibility to disease, or in response to a particular drug treatment. In general, genetic variation has subtle effects on susceptibility to common diseases such as coronary heart disease or diabetes: many genes are involved, and they interact both with one another and with environmental and lifestyle factors to influence whether and when disease will develop. Some sets of these gene variants may cluster in a particular family, so that a family history of disease may be apparent. In rare cases, however, a change (or “mutation”) in a single gene may have a drastic effect, largely over-riding any environmental effects, or effects of other genes, and making disease virtually certain. Examples of such highly “penetrant” single-gene diseases, which show clear inheritance patterns within families, include cystic fibrosis and Huntington’s disease. There are also some rare subsets of common diseases such as breast and bowel cancer that are caused by mutations in single genes (Miller and Kumar, 2001).

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Single Gene Disease

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6.5 Human Genome Project and Health Services As detailed information emerges about the sequence of the DNA and the expression and properties of the resulting proteins we are building up a greater understanding of biological mechanisms and the ways in which they can go wrong in human disease. Increasingly, diseases may no longer be classified by clinical criteria, but on the basis of their underlying molecular pathology. Together with this new molecular understanding may arise new options for prevention and treatment. Disease prevention relies on an understanding of the factors that underlie health and disease (Khoury, 2003). The old dichotomy between “genetic” and “environmental” disease has now been replaced by the realisation that both genetic and environmental factors are at work in almost all diseases. Thus, an important theme of health promotion will be to identify the modifiable risk factors for disease that interact with genetic variation and may be used to target preventive action. In terms of what we can currently do about prevention or promotion of public health, however, we can distinguish between the single gene disorders, where there are even now some clinical applications, and those diseases in which the genetic and environmental interaction is much more complex. We now know that genetic predisposition has an effect in almost all diseases. Diseases with a known genetic component include most of the common diseases such as diabetes, stroke, coronary heart disease, cancers, dementia, depression, and many others – all major contributors to the morbidity and mortality of the population. However, the way in which genetic and environmental factors interact to cause disease is highly complex. Much work will be required to unravel these complexities and determine appropriate strategies for disease prevention. We need to understand the significance of these various factors in populations and sub-populations. The analysis in large populations of the interaction between genetic variation and environmental factors is the relatively new science of genetic epidemiology. Its study will require new techniques for DNA testing, new statistical techniques for analysing hundreds of thousands of people and the utmost care to protect individual privacy.

6.6 Single Gene Disease With the completion of the human genome project, attention is now rapidly shifting towards the study of individual genetic variation. The most abundant source of genetic variation in the human genome is represented by single nucleotide polymorphisms (SNPs), which can account for heritable inter-individual differences in complex phenotypes. Identification of SNPs that contribute to susceptibility to common diseases will provide highly accurate diagnostic information that will facilitate early diagnosis, prevention, and treatment of human diseases (Suh and Vijg, 2005). Already the genetic cause has been identified for more than 200 single-gene diseases. Many of these are devastating conditions that kill their victims during childhood or early adulthood. For most of these diseases genetics

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has so far, in general, offered only the option of reproductive choice for affected families. The ability to test for conditions such as Duchenne muscular dystrophy either before birth or even pre-implantation has enabled some individuals to avoid the devastating consequences of severe childhood disability and death for their family. Ultimately, families affected by single-gene diseases hope that information about the molecular defect involved will enable effective treatments to be developed. The “Holy Grail” for many families is gene therapy, whereby the defective gene in tissue(s) affected by the disease would be replaced by a normal copy. There has been one notable success of gene therapy, to treat a very rare genetic immunodeficiency syndrome, and there is some promising current research on gene therapy for haemophilia, but gene therapy cures for most single-gene diseases still seem to be some way off. For late-onset single-gene diseases, there are additional considerations. Here, the emphasis falls on predictive testing for adults. If no treatment is available, as in the case of Huntington’s disease with its dreadful neurological and psychiatric symptoms, individuals face grave personal dilemmas in considering whether they really want to know their own future. In some other adult-onset single-gene conditions, there is more incentive for predictive testing. Rare single-gene subsets of two common diseases provide examples where there are real opportunities for prevention. It is important to try to identify individuals carrying these gene mutations because their risk of disease is higher and there are effective interventions to reduce their risk. Strategies for identifying these people usually rely on testing individuals in families that have a strong family history of the disease, or in which one family member is known to carry a causative mutation. Familial hypercholesterolemia (FH), which is one of the most common single gene disorders in the population, is associated with premature development of arteriosclerosis and early onset of coronary heart disease. However, there is now great potential to prevent or even reverse the arteriosclerosis in patients with this condition due to advances in drug therapy, particularly the development of HMG CoA reductase inhibitors (statins). Hereditary Non-polyposis Colorectal Cancer (HNPCC) is a rare single-gene subset of colorectal cancer that is caused by a mutation in any one of several different genes that code for DNA repair enzymes. People who have inherited a mutation may have up to an 80% lifetime risk of cancer. Once identified, they can be offered a programme of bowel surveillance by colonoscopy, which can find and remove early growths and almost completely eradicate this risk. The advances in our knowledge of molecular biology and the Human Genome Project have led to greater understandings of genes and gene alterations. These exciting discoveries have led to new ways of thinking about cancers, tumor growth, and how genetic technology may be harnessed to both understand diagnose and alter the course of cancer therapy. Selectively targeting the molecular/genetic abnormalities which give rise to cancers may ultimately result in less toxic treatment approaches which may affect a variety of issues related to quality of life.

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6.7 New and Individualised Drug Treatments Some of the most immediate ways in which the new genetics resulting from the Human Genome Project will make an impact are through developments in drug treatment. Many of the current problems in drug treatment stem from the variable response of patients. This affects efficacy (whether the drug works) and safety (whether or not it produces adverse effects). These differences depend on individual variation in the way in which drugs are absorbed and handled within the body as well as different reactions at target sites. This field of work is known as pharmacogenetics. It is expected that within the next few years there will be many individually tailored prescribing guidelines (Phillips et al., 2001). Drug design will also be revolutionized as researchers create new classes of drugs based on a reasoned approach to data from gene sequencing and protein structure rather than by the traditional trial and error method. Greater knowledge of the human genome will eventually lead to increasing possibilities to predict future health. However, some of the personal and societal consequences are uncertain. We do not know how people might behave in the face of more precise information about their individual risk. Some may not want the information. Some people may react to the knowledge that they are susceptible to a particular disease by taking every precaution, whilst others may regard disease as pre-ordained and feel that nothing they do will make any difference. The question of discrimination may also arise. People worry that their genetic information may be made available to others, their insurer or employer, for example. The arguments about this issue are very complex, but it is important that rational policies are in place to avoid the possibility of discrimination and the danger of creating a “genetic underclass”. These are examples of the ethical, legal and social issues which arise in associations with the new genetics and which are being explored in detail by such bodies as the Human Genetics Commission.

6.8 The Achievements and Challenges of the HGP In 1995, for example, the highest resolution physical maps for human chromosomes were completed by the Los Alamos National Laboratory and the Lawrence Livermore National Laboratory for chromosomes 16 and 19, respectively. The chromosome 19 map has already contributed to the characterization of the genetic defect underlying the disease of myotonic dystrophy and to the description of the unusual genetic mechanism of aberrant triplet repeats that is now known to contribute to the onset of at least nine diseases, including Huntington’s disease. Genes mapped to chromosome 16 include those involved in Batten’s disease, polycystic kidney disease, Crohn’s disease, forms of breast and prostate cancer (Miki et al., 1994), and Fanconi’s anemia, as well as many others. In addition, the DNA repair genes HHR23A, XRCC1, and ERCC2, as well as genes involved in olfactory receptors, Alzheimer’s disease, and one form of migraine headache, have been discovered on chromosome 19.

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Fig. 6.2 The outcomes of the Human genome project allows us to unravel some of the mysteries of life

Lee Hood at the University of Washington in Seattle has sequenced nearly 700,000 consecutive base pairs of DNA from the human T cell receptor complex, as well as a comparable amount of mouse T cell receptor DNA. These sequences have provided surprising insights into the evolution and function of certain white blood cells important for many immune responses to invading pathogens and are widely thought to be involved in both autoimmune diseases and protection against early tumor development. The Human Genome Project has catalyzed the emergence of a new approach to biology termed systems biology (Fig. 6.2). Systems biology analyzes all the interrelationships of the elements in a biological system, rather than studying them one at a time, as has been the modus operandi in biology for the past 30 years. This systems approach has also emerged in the context of the view biology is an informational science and the development of high-throughput tools for capturing biological information and powerful new computational tools for analyzing it. The application of systems approaches to medicine will lead to the rise of predictive, preventive, and personalized medicine over the next 15–20 years, giving rise to a total transformation in how medicine is practiced. Additional accomplishments include advances in technologies that are speeding up DNA sequencing, among them the development of novel “vectors” (critical for the manipulation of DNA in fragment sizes that can readily be characterized and studied), particularly Bacterial Artificial Chromosomes or BACs. A DOE-funded BAC library contributed to the discovery of the Breast Cancer-1 gene by supplying the particular BAC containing the appropriate DNA fragment from chromosome 17.

6.9 Information Transfer To take full advantage of the wealth of information generated by the Human Genome Project, epidemiologists and genetic epidemiologists must have the

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ability to easily access the data, as well as a working knowledge of the retrieval process to properly query, analyze, and interpret the desired information. A primary component of the genome initiative is the development of comprehensive computer databases to assimilate the tremendous amount of mapping and DNA sequence data and to provide links to the scientific and medical literature. Numerous databases have been established to provide organized storage and efficient dissemination of the genome mapping and sequencing data. The informatics movement has generated global computer networks with on-line access over the internet that permit remote access and retrieval of raw or computed data. The seemingly unlimited potential of this technology is readily available to epidemiologic researchers who may be unfamiliar with genetics or genome informatics through simplified accession programs and database helplines. These databases are invaluable to epidemiologic research and should be familiar to all genetic epidemiologists because they contain vast amounts of information regarding the genes and molecular defects that contribute to human disease, methods for rapid detection of mutations and polymorphisms (if available), comprehensive descriptions of disease phenotypes, and the status of treatment and intervention strategies.

6.10 Impact of the Human Genome Project on Epidemiologic Research The discipline of genetic epidemiology has greatly expanded the applicability and utility to the public of genetic advancements, including the mapping of genes responsible for Mendelian diseases (such as cystic fibrosis) and the development of models to predict disease (such as the multiple-step mechanism carcinogenesis). The greatest challenges confronting genetic epidemiology, however, are the common chronic diseases with late age-of-onset which exert a tremendous burden on public health as measured by morbidity, mortality, and cost. The greatest impact and benefit to public health from genetic epidemiologic research will likely come from uncovering and better understanding the genetic etiology of the common chronic diseases (such as coronary artery disease and diabetes) and the common forms of cancer (such as breast and colon cancer). Until recently, genetic epidemiology made inferences primarily from statistical analyses of the distribution of disease or other traits among family members. Direct measures of genetic information were rare and, with the exception of the human leukocyte antigen (HLA) complex, were limited to red cell antigens and polymorphic red cell and plasma enzymes. For the field of genetic epidemiology to achieve its full potential and better characterize the genetic etiology of the common chronic diseases, high quality genetic markers were necessary for gene mapping, and improved methods needed to be developed to detect and quantify functional alleles (Goldstein et al., 1973). The Human Genome Project and other developments in molecular biology are providing the necessary tools for epidemiology and genetic epidemiology to uncover the molecular mechanisms for variation in the distribution of disease among families and populations. Although there have been other conceptual and technical advances

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(particularly in the area of computational methods) that have advanced the field of genetic epidemiology.

6.11 Impact of the Human Genome Project on Our Genomic Makeup The Human Genome Project has already fuelled the discovery of more than 1,800 disease genes (Venter et al., 2001). As a result of the Human Genome Project, today’s researchers can find a gene suspected of causing an inherited disease in a matter of days, rather than the years it took before the genome sequence was in hand. There are now more than 1,000 genetic tests for human conditions. These tests enable patients to learn their genetic risks for disease and also help healthcare professionals diagnose disease. The Human Genome Project (HGP) has brought unprecedented insight into our genomic makeup (Venter et al., 2001). Genomic diagnostics makes practical use of the wealth of HGP data by determining correlations between clinical phenotypes and their underlying genotypes, enabling us to give etiological faces to diseases for which none existed before. An area where genomic diagnostics is anticipated to have great impact is prenatal testing. Because so much remains unknown about what causes spontaneous abortions or the development of idiopathic mental retardation, the medical community is eager to apply the latest in comprehensive genomic diagnostics to the prenatal setting. However, within medicine, few concerns generate as much anxiety in those it affects – namely, expecting parents – as prenatal diagnostics. This anxiety is justified considering full understanding of the clinical relevance of the HGP data often lags behind the technologies that utilize that information. With complete appreciation for every genotype/phenotype consequence yet to be attained, care should be taken when using this information in the prenatal setting. Additionally, sensitivities from sociological, psychological and ethno-religious perspectives further demands that prenatal genomic analysis be approached with exceptional caution and responsibility. A primary technology that has parlayed the information from the HGP into clinical utility is that of array-based comparative genomic hybridization (aCGH). Because this method makes use of the content of the human genome to screen a clinical sample for genomic copy number changes and hence many disease associations simultaneously in a patient, aCGH has become the most anticipated test in prenatal genetics. Combimatrix Molecular Diagnostics (CMDX) is a pioneer in the conversion of the HGP data into clinical diagnostics. CMDX is leading the vanguard of prenatal testing by offering cutting-edge genomic diagnostics while cognizant of its associated sensitivities and issues, ensuring medical practitioners receive the very best, responsible, and appropriate genomics based prenatal diagnostic testing. And each step of the way, just like in the HGP, new techniques are developed that are applicable to clinical medicine. Our knowledge of human genetics for identifying new genes and understanding how they work increases in unpredictable ways.

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This promises to expand the number and types of clinical laboratory testing that are offered to patients. Rapid discoveries of novel genes for a variety of human diseases are anticipated as genomic maps are- come more detailed and methods for mapping and characterizing disease genes become more refined (Sachidanandam et al., 2001). Understanding the genetics behind ocular disorders opens a number of opportunities to address disease management, not least of which is the development of molecular diagnostics. Molecular diagnostics is being revolutionized by the completion of the human genome project and by the development of highly advanced technologies for DNA testing (Ferrari et al., 2003). One of the most important challenges is the introduction of high throughput systems such as DNA chips into diagnostic laboratories. DNA microchips are small devices permitting rapid analysis of genetic information, exploiting miniaturization of all components and automation of operational procedures. The most important biochip applications include gene expression and genetic variation identification and both may improve human molecular diagnostics. Recent and continuing developments in genome technology and analytic methods provide the tools and raw materials for unravelling the complexities of the common chronic diseases and common forms of cancer. One major step toward such comprehensive understanding was the development in 2005 of the HapMap, which is a catalogue of common genetic variation, or haplotypes, in the human genome. HapMap data have accelerated the search for genes involved in common human diseases, and have already yielded impressive results in finding genetic factors involved in conditions ranging from age-related blindness to obesity.

6.12 Conclusion In the future, it is clear that genetic discoveries, including the vast amount of information being made available by the HGP, will lead to opportunities for treatment. The progress in treatment will tend to reduce the dilemmas surrounding genetic testing. In some cases, the neurologist will still be left with the dilemma of a disease that can be accurately diagnosed but for which no effective therapy is yet available. However, the vast array of biological information made available by the genome project will also allow widespread approaches of emerging technologies such as gene therapy, in which defective gene sequences may be replaced. In the case of other disorders, the knowledge gained by genetic dissection may lead to new, more effective drug therapies or immediate approaches to improving health through altering lifestyle (Evans, 1998). Genome medicine in the next century is likely to take on an entirely new role in society based on the beginnings inherent in the HGP (Nathans and Orkins, 2009). The human genome project was borne of technology, grew into a science bureaucracy in the U.S. and throughout the world and is now being transformed into a hybrid academic and commercial enterprise. The next phase of the project promises

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to veer more sharply toward commercial application, exploiting the rapidly growing body of knowledge about DNA structure to the pursuit of practical benefits. This Project has catalyzed the emergence of a new approach to biology termed systems biology. The application of systems approaches to medicine will lead to the rise of predictive, preventive, and personalized medicine over the next 15–20 years, giving rise to a total transformation in how medicine is practiced.

References Collins, F.S., Patrinos, A., 1998, New goals for the U.S. Human Genome Project: 1998–2003 Science 282(5389):682–689. Evans, G.A., 1998, The Human Genome Project: applications in the diagnosis and treatment of neurologic disease. Arch Neurol 55(10):1287–1290. Ferrari, M., Stenirri, S., Bonini, P., Cremonesi, L., 2003, Molecular diagnostics by microelectronic microchips. Clin Chem Lab Med 41(4):462–467. Goldstein, J.L., Schrott, H.G., Hazzard, W.R., Bierman, E.L., Motulsky, A.G., 1973, Hyperlipidemia in coronary heart disease II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. Clin Invest 52(7):1544–1568. Khoury, M.J., 2003, Genetics and genomics in practice: the continuum from genetic disease to genetic information in health and disease. Gen Med 5(4):261–268. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P.A., Harshman, K., Tavtigian, S., Liu, Q., Cochran, C., Bennett, L.M., Ding, W., et al., 1994, A strong candidate for the breast and ovarian cancer susceptibility gene, BRCA1. Science 266(5182):66–71. Miller, M.P., Kumar, S., 2001, Understanding human disease mutations through the use of interspecific genetic variation. Hum Mol Genet 10(21):2319–2328. Nathans, D.G., Orkins, S.H., 2009, Musing on genome medicine: genome wide association studies. Genome Med 1(1):3 Peltonen, L., 2001, The molecular dissection of human diseases after the human genome project. Pharmacogenome J 1(1):5–6. Phillips, K.A., Veenstra, D.L., Oren, E., Lee, J.K., Sadee, W., 2001, Potential role of pharmacogenomics in reducing adverse drug reactions: a systematic review. JAMA 286(18):2270–2279. Risch, N., Burchard, E., Ziv, E., Tang, H., 2003, Categorization of humans in biomedical research: genes, race, and disease. Genome Biol 3(7):1–12. Sachidanandam, R., Weissman, D., Schmidt, S.C., Kakol, J.M., Stein, L.D., Marth, G., Sherry, S., Mullikin, J.C., Mortimore, B.J., Willey, D.L., 2001, A map of the human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 409(6822): 928–933. Suh, Y., Vijg J., 2005, SNP discovery in associating genetic variation with human disease phenotypes. Mutat Res 573(1–2):41–53. Van Ommen, G.-J.B., 2002, The Human Genome Project and the future of diagnostics, treatment and prevention. J Inher Metab Dis 25(3):183–188. Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O., Yandell, M., Evans, C.A., Holt, R.A., et al., 2001, The sequence of the human genome. Science 291(5507):1304–1351.

Chapter 7

Molecular Diagnosis in the Post Genomic and Proteomic Era

Abstract The completion of the human genome project is widely regarded as a turning point in biology and medicine. As we usher in the “post-genomic” era, we are faced with an explosion of new information, which is leading to dramatic changes in the way we are able to study and manipulate life. Over the last few years, bimolecular research has progressed to functional genomics and the application of this knowledge to advance our understanding of health and disease. It is clear that genomic information alone is not sufficient to completely explain disease states. Post-genomic approaches attempt to contribute to our understanding of this interaction, with each approach capturing a different angle of the global picture. Intuitively, the next step forward is to integrate these datasets, an approach that, if successful, could be much more informative and predictive than working exclusively on a single platform. Keywords Genomics · Transcriptomics · Proteomics · HGP · Biomedical research · Drug discovery · Sequencing · Functional genomics · Microarrays · Environmentome · Multigenetic diseases · Candidate markers · Personalized medicine

7.1 Prologue The year 2003 marked the completion of the first draft of the human genome sequence. We now find ourselves in the modern genetic era, trying to understand and utilize a previously unimaginable amount of biological information. Scientists have the daunting task of finding new ways to use the human genome sequence to improve drug discovery and development efforts and to find better ways to combat disease for the benefit of humanity (Peltonen and McKusick, 2001).

7.2 The Genomic Era The genomic era has brought with it a basic change in experimentation, enabling researchers to look more comprehensively at biological systems. The sequencing of the human genome coupled with advances in automation and parallelization M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_7,

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technologies have afforded a fundamental transformation in the drug target discovery paradigm, towards systematic whole genome and proteome analyses. In conjunction with novel proteomic techniques, genome-wide annotation of function in cellular models is possible. Overlaying data derived from whole genome sequence, expression and functional analysis will facilitate the identification of causal genes in disease and significantly streamline the target validation process. Moreover, several parallel technological advances in small molecule screening have resulted in the development of expeditious and powerful platforms for elucidating inhibitors of protein or pathway function. Conversely, high-throughput and automated systems are currently being used to identify targets of orphan small molecules. The consolidation of these emerging functional genomics and drug discovery technologies promises to reap the fruits of the genomic revolution. In 1990, the human genome project was established to sequence the human genome (Collins et al., 2003), with the aim of applying the acquired genomic data to improve disease diagnosis and determine genetic susceptibility. The publication of the first draft sequence of the human genome in 2001(Venter et al., 2001) was thus followed by a rapid growth of different approaches to extract useful information from the genomic sequence. Following the publication of the complete human genomic sequence, the post-genomic era is driven by the need to extract useful information from genomic data (Tang et al., 2009). What is now to be done is to work out the function of the genes. Hence, the post-genome era has been called functional genomics, which includes proteomics, the technology and strategies required to determine the function of proteins. How this will be accomplished remains to be determined, but new technologies will be needed. The use of microarrays (a method by which the expression of many thousands of genes can be identified very rapidly with microchips) is an early, promising strategy in functional genomics. Bioinformatics will need to come up with more sophisticated programs by which the function of genes can be predicted. The traditional “wet-lab” approach to research might even give way to a complete “in-silico” (i.e., computer) strategy! The first challenge – sequencing the genome – has been accomplished. Many more even larger challenges await us as we set out to determine the function of all human genes. The next decade will see the study of the transcriptome representing all of the transcripts or RNA copies of the genes in a cell; proteome, representing all of the proteins in a cell, tissue or individual; and metabolome, representing all of the molecular components of a cell or tissue produced by the proteins (Venter et al., 2008).

7.3 Concept The molecular diagnostics industry is experiencing explosive growth. Innovation and strategic planning is needed to realize the commercial potential from applied genomics. The number of product offerings for molecular profiling that allow the early detection, prognosis, and treatment of disease are increasing substantially. But the advance of companion diagnostics has been slow to take hold.

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Molecular diagnostics is here to stay. Not only are molecular diagnostic techniques being developed for new tests as new disease genes are being discovered, but molecular diagnostic techniques also are being developed that replace traditional tests, especially in the areas of bacteriology and virology. Molecular diagnostics may provide several clinical advantages including the production of a clear unequivocal test, the provision of critical information to inform genetic counseling, the establishment of prenatal screening services and the identification of individuals at a pre-symptomatic stage so that environmental factors, if relevant, may be adjusted. The ultimate goal, of course, would be to design therapeutics based on the genetic information. Perhaps the greatest utility of the vast genetic information being generated by the human genome initiative is in primary prevention programs. Methods for identifying asymptomatic individuals at risk for genetic disease and the development of more efficacious intervention strategies are becoming paramount as health care costs escalate and medical genetics shifts to early detection and prevention of disease. With these abilities will come the need to fully integrate genetic information into large prospective studies, and intervention trials to accurately predict disease risk and synthesize new approaches to risk reduction. Understanding the role of genes in human disease will improve our understanding of genetic disease etiology as well as our ability to predict disease. Insight into the genetic basis of chronic disease etiology will have immediate impact by suggesting novel therapeutic approaches and aiding new drug discovery. At least 350 biotechnology-based products resulting from the Human Genome Project are currently in clinical trials. Having the complete sequence of the human genome is similar to having all the pages of a manual needed to make the human body. The challenge now is to determine how to read the contents of these pages and understand how all of these many, complex parts work together in human health and disease. The wait is over, and the post genomic era has arrived. Genomics, transcriptomics, proteomics, metabolomics, epidemiological data and microbial data provide different angles to our understanding of gene-environment interactions and the determinants of disease and health. Our goal and our challenge are to integrate these very different types of data and perspectives of disease into a global model suitable for dissecting the mechanisms of disease and for predicting novel therapeutic strategies. We are in for a veritable revolution, the diagnosis of infections, development of vaccines, new treatments for old bacteria, predictive medicine, and innovative therapies. A truly amazing future awaits us (Fig. 7.1). What is now required is that more training in genetics be imparted and more genetic centers be opened so that the tremendous benefits of this technology are utilized for the benefit of many patients in India now and in future. A similar transition has occurred in infectious disease following the publication of complete genome sequence for Mycobacterium tuberculosis (Young, 2001), Vibrio cholera, and at least 30 other microbial species. Genetic material isolated from pathogenic organisms in ancient bone tissue or herbarium samples collected from 1845 through 1847 during the Irish potato famine has been used to characterize the infectious agent using polymerase chain reaction or sequencing of ribosomal

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Fig. 7.1 The challenges of the post genomic era

DNA and mitochondrial DNA. It will now be possible to integrate bacterial post genomic data with human post genomic data for historical and current investigation into the pathogenesis of infectious disease, antibiotic resistance, and epidemiology (Raczniak et al., 2001). Each of the post-genomic approaches has already contributed to our understanding of specific aspects of the disease process and the development of diagnostic/prognostic clinical applications (Scuturi et al., 2007; The wellcome trust case control consortium, 2007). Cardiovascular disease (Edward et al., 2008 ; Giovane et al., 2008), obesity (Blakemore and Froguel, 2008; Loos et al., 2008; Pietilainen et al., 2007), diabetes (Chen and Hess, 2008; Bougneres and Valleron, 2008; Orešic et al., 2008; Frayling, 2007), autoimmune disease (Su, 2008) and neurodegenerative disorders (Quintana et al., 2008; Papassotiropoulos et al., 2005) are some of the disease areas that have benefited from these types of data. Taking the metabolic syndrome as an example, our knowledge on all aspects of the disease has grown. The metabolic syndrome is the result of a complex bioenergetics problem characterized by disturbances in lipid, carbohydrate and energy metabolisms and blood pressure. In combination, these metabolic factors contribute to an increased susceptibility to cardiovascular disease, morbidity and mortality (Cornier et al., 2008). Genome-wide association (GWA) studies have identified possible genes involved in each aspect of the syndrome: namely type 2 diabetes (Frayling, 2007), obesity (Lindgren and McCarthy, 2008) and hyperlipidaemia (Hegele, 2009). The findings have confirmed the role of certain candidate genes as well as the polygenetic nature of the syndrome. Not surprisingly, replicate GWA studies of type 2 diabetes revealed that the genes associated with disease, among others, are involved in beta-cell function and adipocyte biology (Frayling, 2007; Lindgren and McCarthy, 2008; Perry and Frayling, 2008). In contrast, genes found to be associated with obesity appear to be those that are predominantly involved in central appetite regulation (Li and Loos, 2008) as key contributors to positive energy balance. Genetic association studies in epidemiology have highlighted a number of issues. Firstly, many common disease states are related to either many genetic polymorphisms of small effect or, in selected cases, to a few of large effect. The involvement of multiple genes with unequal contributions to disease hints of complex gene-gene and gene-environment interactions. The understanding of such interactions becomes

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a daunting task when other modulating factors remain unknown. Secondly, some common diseases such as type 2 diabetes appear to be relatively less genetically determined compared to diseases such as rheumatoid arthritis and obesity (Maes et al., 1997). In these situations, our understanding of pathophysiology requires additional data outside of genomic information. Thirdly, the initial failures to find robust replicable associations between most of the identified genetic variants and common complex diseases suggest that genomic analysis alone will not account for all of the heritability and phenotypic variation (Maher, 2008). For this reason, there is a growing need to incorporate information derived from environmental studies and post-genomic data into genetic analysis.

7.4 The Post Genomic Era In the post-genomic era of biology, focus is slowly shifting from the raw sequence data generated from these large multinational projects, to downstream consequence of that sequence, such as gene function, and its relationship to phenotype. As such, the “omics” technologies are enjoying a boom. Transciptomics, the study of gene expression, proteomics, the study of protein expression, and metabolomics, and the snap-shot observation of metabolic status are of great current interest. The new science of proteomics promises exciting insights into the working of the cell. Such technologies offer a powerful insight into molecular mechanisms, and have great utility in functional genomics and diagnostics, to name but two applications. A common technological approach for the studies of proteomics and metabolomics is mass spectrometry, which in combination with chromatography can detect, identify and quantify large numbers of metabolites from a single sample. One of the greatest challenges in modern medicine is to dissect the cascade of molecular events that lead to the development and progression of tumours. As we emerge into the post-genome era, proteomics finds itself as the driving force field as we translate the nucleic acid information archive into understanding how the cell actually works and how disease processes operate. The genome-wide study of proteins embodies the exciting promise of proteomics (Mocellin et al., 2004). Even so, the traditionally held view of proteomics as simply cataloging and developing lists of the cellular protein repertoire of a cell are now changing, especially in the sub-discipline of clinical proteomics. The most relevant information archive to clinical applications and drug development involves the elucidation of the information flow of the cell; the “software” of protein pathway networks and circuitry. The deranged circuitry of the cell as the drug target itself as well as the effect of the drug on not just the target, but also the entire network, is what we now are striving towards. Clinical proteomics, as a new and most exciting sub-discipline of proteomics, involves the bench-to-bedside clinical application of proteomic tools. Unlike genomics, proteomics aims ambitiously to study not only protein expression profiles but also protein functions, which should provide researchers with a more comprehensive view of the molecular machinery that governs cellular functions (Chanda and Caldwell, 2003).

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Unlike the genome, there are potentially thousands of proteomes: each cell type has its own unique proteome. Moreover, each cell type can alter its proteome depending on the unique tissue microenvironment in which it resides, giving rise to multiple permutations of a single proteome. Since there is no polymerase chain reaction equivalent to proteomics, identifying and discovering the “wiring diagram” of a human diseased cell in a biopsy specimen remains a daunting challenge. New micro-proteomic technologies are being and still need to be developed to drill down into the proteomes of clinically relevant material. Cancer, as a model disease, provides a fertile environment to study the application of proteomics at the bedside. The promise of clinical proteomics and the new technologies that are developed is that we will detect cancer earlier through discovery of biomarkers, we will discover the next generation of targets and imaging biomarkers, and we can then apply this knowledge to patient-tailored therapy (Krieg and Paweletz, 2002). Cancer proteomics is an exciting field that is witnessing many new developments in recent years. It is hoped that these advances will result in decreased cancer death rates, which have not declined dramatically in the last several decades. Some of the problems with current tumor markers include the lack of sensitivity and specificity, factors that prevent their use in population-based screening of disease. Thus, there is an urgent need to identify novel biomarkers that can faithfully detect the disease state. As we are now in the post-genome era, many opportunities have been created. Genomic sequence data are available for human, as well as several other species. We are now poised to mine these data and to determine the functions of the encoded proteins constituting the human genome. Proteomics affords this opportunity by providing enhanced procedures and tools for discovery and also a framework for understanding these components in terms of pathogenesis. New technologies and improvements in existing methodologies will allow for the rapid growth in the identification and characterization of peptides and proteins that are unique to various clinical states. This technology can be successfully applied to clinical specimens for the identification of disease state.

7.5 Advantages of Combining Multiple Types of Data It is clear that the genetic approach captures only one layer of the complexity inherent within human biology. There is thus a need to integrate multiple “omics” datasets when aiming to unravel the molecular networks underlying common human disease traits (Zhu et al., 2007). Attempts have been made to combine two datasets in relation to the clinical phenotype, and this is reflected in the combination of terms found in the literature, for example metagenomics, pharmacogenomics and epigenetics. Many of the post-genomic approaches linking the genetic association data with other “omics” layers focus on the use of “omics”- derived phenotypic data as quantitative traits. The utility of such approaches has been previously applied, by combining genetics and metabolomics, in plant functional genomics (Raamsdonk et al., 2001). More recently, such approaches have also been applied to human

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datasets. For example, Papassotiropoulos and colleagues (2005) identified clusters of cholesterol-associated susceptibility genes for Alzheimer’s disease by combining genetics with sterol profiling, while Gieger and colleagues (2008) used ratios of metabolites to identify the function of putative genes. In another study, proteomics was linked to quantitative trait loci (QTL) in an attempt to identify changes in function rather than quantity of the protein (Stylianou et al., 2008). By combining multiple types of techniques, including genetics, transcriptomics, proteomics and metabolomics, we are expecting a shift toward “environmentome” research, where all available information from preconception to disease onset, using both longitudinal and cross-sectional experimental designs, can be obtained (Bougneres and Valleron, 2008). The measurement of traits that are modulated but not encoded by the DNA sequence, commonly referred to as intermediate phenotypes, is of particular interest. These intermediate phenotypes include not only biochemical (metabolites) and genomic (gene expression) traits, but also an individual’s microbial (gut microflora) (Turnbaugh et al., 2007; Turnbaugh and Gordon, 2008) and social traits. It is conceivable that by comprehensively examining an individual’s “environmentome”, we would be able not only to understand both the genetic and environmental determinants of disease, but also to develop “feasible” personalized medicine, that is, tailor specific personalized interventions to the individual’s own environmental profile. As a pioneering example of this kind, Orešic Land and colleagues (2008) investigated metabolic profiles of children between birth and type 1 diabetes onset in a large birth cohort, and established that specific metabolic phenotypes, not dependent on human leukocyte antigen (HLA)associated genetic risk, precede the first autoimmune response. The excitement of this research is the expectation that these early metabolic phenotypes may be validated as specific diagnostic and prognostic markers of disease, with therapeutic implications. The goal of inferring disease causality and disease mechanisms from integrated data is complicated by the fact that measuring more variables may provide a better characterization of the process but still does not contribute directly to our understanding of cause and effect. In fact, given the progressively increasing number of variables that we can measure, the odds of finding spurious associations that do not reflect true causality are much higher. Confounding and reverse causality are among the main sources of bias for failures to replicate apparently robust associations between risk factors and diseases (Lawlor et al., 2006). Confounding specifically refers to a spurious causal effect inferred from the association between a risk factor and a disease due to the existence of some common causes, that is, confounding factors to both of them. This type of spurious causal effect can be removed if we have enough knowledge about the most likely confounding factor candidates. However, the truth is that for most epidemiological studies confounding factors are unknown and difficult to measure, especially in case-control studies. Reverse causality, the second source of bias, refers to an alternative explanation for the observed association between a risk factor and disease, which states that the “risk factor” is a result of the disease, rather than vice versa. The problem of reverse causality is particularly prevalent in retrospective case-control studies.

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One example of a potential confounding association is the established epidemiological evidence of a strong link between obesity and insulin resistance. This association has recently been brought into question from the identification of specific clinical settings where fat mass dissociates from insulin resistance (Garg, 2004; Wildman et al., 2008). This implies that adipose tissue expansion typically associated with obesity per se may not be the cause of metabolic complications. A potential alternative explanation may be related to an individual’s ability to optimally store fat. In the presence of caloric excess, a person is likely to remain metabolically healthy despite obesity, provided their adipose tissue can continue to expand and safely store fat. Therefore, while the epidemiological evidence associates the risk of metabolic complication with increased body weight, this relationship may not be direct and may not necessarily reflect a truly biologically relevant process.

7.6 Biomedical Research in the Postgenomic Era The genomics era has provided great opportunities for deciphering the genes that are mutated in human diseases. Identification of a causative pathogen is essential for the choice of treatment for most infectious diseases. Many FDA approved molecular assays; usually more sensitive and specific compared to traditional tests, have been developed in the last decade. A new trend of high throughput and multiplexing assays are emerging thanks to technological developments for the human genome sequencing project. The enthusiasm for the introduction of genetic screening for single-gene or multigenetic diseases is great in the medical community. However, in general, a reasonable number of individuals need to be screened to prevent one case of disease. Prevention is achieved only by screening for highly penetrant mutations in high-risk families and not for such mutations in the general population or for low-penetrant polymorphisms. The frequency with which the phenotype the gene controls is seen in individuals who carry it describes the penetrance of the gene. The differences observed in the penetrance of a mutation depend on 6 factors (Vineis et al., 2001) role of the protein encoded in metabolism, functional consequence of the mutation, gene-gene interactions, onset of somatic mutations, environmental interactions, and the presence of alternative metabolic pathways that can correct the loss of function caused by the mutation. Proteomics technologies are now offering unique chances to identify new candidate markers. In the diagnostic and the pharmaceutical industry there is a constant need for new diagnostic markers and biomarkers with improved sensitivity and specificity. During the last 5 years, only a few novel diagnostic markers have been introduced into the market. Before a marker can be introduced into the market, three successive developmental phases have to be completed: the discovery phase, in which a variety of proteomics technologies are applied to identify marker candidates; the prototype developmental phase, in

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which immunological assays are established and validated in defined sample collectives; and finally the product development phase, with assay formats suitable for automated platforms. The hurdles that a potential candidate marker has to pass in each developmental phase before reaching the market are considerable. The costs are increasing from phase to phase, and in industry a number of questions concerning the medical need and the potential return on investment have to be answered before a proteomics discovery project is started. As the biomedical research community enters the post-genome era, studying gene expression patterns and phenotypes in model organisms will be an important part of analyzing the role of genes in human health and disease. New technologies involving DNA chips will improve the ability to evaluate the differential expression of a large number of genes simultaneously. Also, new approaches for generating mutations in mice will significantly decrease the cost and increase the rate of generating mutant lines that model human disease (Woychik et al., 1998). Personalized medicine will provide the link between an individual’s molecular and clinical profiles, allowing physicians to make the right patient-care decisions and allowing patients the opportunity to make informed and directed lifestyle decisions for their future well-being. Clinical diseases as we know them will be replaced by molecular classification. Therapies directed at the root cause of disease will replace those that simply treat the symptoms of disease. Finally, a pharmacogenomic test that predicts therapy response based on a patient’s genomic profile will accompany many drugs. Personalized medicine will involve radical changes in the pharmaceutical industry and medical practice and is likely to affect many aspects of society. Most importantly, the individual whose health is at stake will benefit enormously. The advent of disease gene patents threatens to constrain the medical community’s ability to provide the appropriate standard of care (Balter, 2001). Such a patent has the potential of monopolizing medical testing services and potentially leading to restrictions on research, conflicts of interest, and restricted access to testing services (Merz, 1999). In the clinical laboratory, DNA or RNA represents the most common starting material for molecular-based assays. Research in the post genomic era will focus on identifying the critical messenger RNAs (mRNAs) (transcriptomics) or proteins (proteomics) and the sequence of interactions required to induce a specific biochemical event. Microarrays or “lab-on-a-chip” may contain a library of genes associated with a specific metabolic pathway that are immobilized in a grid (Lockhart and Winzeler, 2000). The post genomic era will present numerous technological and intellectual challenges. The post genomic era will challenge our traditional problem-solving methods and require more creative, efficient, and accurate techniques for characterizing the clusters of nucleotides within genes and associating their mutation with phenotype and disease treatment. Mitochondria play a central role in the intracellular signal transduction pathway, leading to apoptosis or programmed cell death (Gottlieb, 2000). This genetically controlled process regulates tissue size in opposition to mitosis. It is characterized by

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cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation into 180– to 200–base pair units and multiples of these units, and phosphatidylserine translocation and activation of the apoptotic signal transduction pathway. This pathway may represent the final common pathway for cell death in neurodegenerative disease (Offen et al., 2000), which is included in diseases characterized by increased apoptotic activity. The treatment of human disease by delivering genes to human tissues is called gene therapy. Applications in cardiovascular disease include ischemia, restenosis, graft failures, arterial cytoprotection, atherogenesis, and thrombogenesis (O’Brien and Simari, 2000). However, efficacy and toxicity have slowed progress in this field. Complications have included inefficient gene expression and adverse inflammatory reactions. However, encouraging results have been reported in ischemia by the introduction of vascular endothelial growth factor or fibroblast growth factor genes. When these genes are placed in cardiac tissue adjacent to an occluded coronary artery, they stimulate new blood vessel growth (angiogenesis). A different vector was used by Roth et al., 2001 to deliver the factor VIII gene to patients with hemophilia A. They transfected skin fibroblasts from the patient with a plasmid containing factor VIII and injected these cells. Although the results were encouraging, not all patients responded with reduced bleeding and increase in plasma factor VIII activity. Resolution of the problems related to vectors for gene transfer will accelerate investigation in this area. Moldovan and Ferrari describe a silicon-based angiogenesis-assisting device or “angiochip” to release multiple growth factors and other biochemicals to support the growth of endothelial cells grown on the surface. This device would initiate new blood vessel growth without the need for gene therapy.

7.7 Conclusion The post-genome era has begun, and with it the promise of tailoring the practice of medicine to the individual. This emerging field of personalized medicine encompasses the use of risk algorithms, molecular diagnostics, targeted therapies and pharmacogenomics to improve health care. From SNP maps to individual drug response profiling, the human genome sequence will lead to improved diagnostic testing for disease susceptibility genes and individually tailored treatment regimens for those who have already developed disease symptoms. Molecular diagnostics, the use of DNA-, protein- or mRNA-based biological markers to predict the risk of developing disease or the molecular phenotype of an existing one, will change the way we currently define disease. Genomic analysis of diseases with homogeneous clinical phenotypes will unveil distinct molecular entities that require different treatment strategies for optimal outcomes. The completion of the genome sequence is not the beginning of the end, but is only the end of the beginning.

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Chapter 8

Ethics, Patents and Regulations

Abstract The ethical, legal, and social implications (ELSI) program was created by the HGP to address issues that arise as a result of the program but that are not directly related to sequencing of the genome. Clinicians, scientists, patient advocacy groups, and government agencies worldwide are debating the optimal legal protections to prevent abuse. Massive amounts of data flowing from the Human Genome Project and other genomics projects have stimulated an avalanche of applications for patents on genes and gene fragments. Keywords Ethics · Patents · Human genome project · Genetic testing · Diagnostics · Genetic assay · SNP · Single nucleotide polymorphism · Germ line therapy · Somatic gene therapy · Genetic testing · Genomic patents · Expressed sequence tags (ESTs) · Genome technology · Genetically modified organism · HGP

8.1 Prologue The impact of commercialization and patenting pressure on genomics research is still a topic of considerable debate in academic, policy and popular literature (Murdoch and Caulfield, 2009). Recent advances in molecular genetics challenge the scientific community to understand and implement genetic knowledge. Despite excitement about the potential benefits of new genetic information, concerns have been raised about the inappropriate use of genetic testing, clinicians’ incorrect ordering and misinterpretation of test results, and discrimination in employment and insurability based on tests results. Among the public there is fear and mistrust, in part based on horrifying historical events that were gross violations of medical ethical standards. Knowledge of a patient’s genetic profile brings great responsibility and many concerns. Who has the right to access this information? How should (or shouldn’t) it be used?

8.2 Concept of Ethical, Legal, and Social Issues The advances in the field of molecular biology and genetics have widened the possibilities for the diagnosis and treatment of hereditary diseases. At the same M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_8,

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time research into this field has broken bounds of its legal and ethical regulation (Gutiérrez-Samperio, 2002). Ethical issues are generally defined as those raising questions concerning what is moral or right. Legal issues are those concerning the protections that laws or regulations should provide. Social issues are concerned with how events may affect society as a whole and individuals in society. Clearly, these aspects of the Human genome project and its possible outcomes are not independent of each other. The programs focus on ethical, legal, cultural, social, and psychological consequences that could affect policy. The project’s goals included not only identifying all of the genes in the human genome, but also to address the ethical, legal, and social issues which might arise from the availability of genetic information (Durfy, 1993). Five percent of the US annual Human Genome Project (HGP) budget was allocated to address the Ethical Legal Social Imlications (ELSI) arising from the project. Still, many issues, such as the confidentiality of a patient’s genetic profile, remain unaddressed by legislation. In addition to providing guidelines for potential new policy, the program examines philosophical, theological, ethical and other social issues in order to ease the entry of genetic information into clinical practice as well as the social consciousness.

8.3 Ethical and Social Issues in Diagnostic Molecular Genetics Prior to the genome initiative, ethical issues in molecular genetics focused primarily on monitoring and regulating experimentation in recombinant DNA and genetic engineering. Organizers of the Human Genome Project (HGP) understood from the beginning that the scientific activities of mapping and sequencing the human genome would raise ethical, legal, and social issues that would require careful attention by scientists, health care professionals, government officials, and the public (Fig. 8.1). The Ethical, Legal, and Social Issues (ELSI) Program has been established to examine various issues associated with the generation and dissemination of a vast array of genomic information (Meslin et al., 1997; Zneimer, 2002). Highpriority issues initially targeted by the Ethical, Legal, and Social Issues Program for development of policies and guidelines included: 1. The integration and impact of new genetic tests and the debate over population screening; 2. Privacy and confidentiality of genetic information; 3. Genetic counseling and reproductive decisions influenced by diagnostic results; and 4. Public education. In recent years, the Ethical, Legal, and Social Issues Program has also emphasized technical problems such as the potential for genetic discrimination, educating physicians in the advantages and limitations of genetic data, quality control in DNA testing laboratories, and defining guidelines for obtaining informed consent

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Fig. 8.1 Ethical issues before and after HGP

for genetic research. In addition, these groups are developing clinical guidelines for optimal use. Traditional ethical and legal standards of confidentiality between physicians and their patients are under scrutiny. A new principle, “the duty to warn,” is emerging that has applications specific to genetic testing and may conflict with the duty to maintain patient confidentiality. Emerging ethical, legal, and social issues involve the appropriate use and protection of confidential data in tissue and serum banks. Education of the profession and the public at many levels will increase the likelihood that the unraveling of the human genome will maximally benefit society. If fear of genetic testing can be alleviated, selection bias in research could be reduced. Professional and lay organizations concerned with disease should consider a more active role in the public and professional debate, and foster education at all levels. As new genetic assays are introduced into clinical practice, rigorous adherence to established protocols and quality control assurance are of paramount importance. Attention must be directed toward the debate over implementing population-wide screening programs as routine practice in clinical medicine to detect those at increased genetic risk. The increasing ability to diagnose individuals at risk for genetic diseases for which there are no therapeutic options will require enactment of measures to (1) prevent insurance and/or employment discrimination against asymptomatic carriers and (2) accommodate the psychological needs of those who are likely to develop a late-onset condition. Improvement of noninvasive prenatal and preimplantation diagnostic procedures is creating an immediate need to explore the ethical dilemmas and difficult reproductive choices faced by prospective parents known to carry disease-associated genes.

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Increasing public awareness of their availability, benefits, and limitations of molecular diagnostic tests is anticipated to simultaneously improve health care delivery while minimizing the potential for psychological and social stigmatization. The impending explosion in the number of well-characterized human disease genes and new abilities to diagnose genetic disorders will likely necessitate development of novel avenues for education and genetic testing (McCunney, 2002). Careful integration of genetic information allow a practical system for characterizing and resolving ethical and social issues and provide future directions for the fields of molecular genetics and clinical medicine.

8.4 Confronting the Ethical, Legal and Social Issues James Watson, who won the Nobel Prize in Physiology in Medicine in 1962 for codiscovering the structure of DNA, made a seminal contribution to the HGP when he recognized that knowledge derived from genome studies has broader medical and societal implications. This led directly to the establishment of a program devoted to the ethical, legal, and social implications (ELSI) of genome research. The ELSI program (Masui and Takada, 2003) focused on the possible consequences of genomic research in four main areas: • Privacy and fairness in the use of genetic information, including the potential for genetic discrimination in employment and insurance. • The integration of new genetic technologies, such as genetic testing, into the practice of clinical medicine. • Ethical issues surrounding the design and conduct of genetic research with people, including the process of informed consent. • The education of healthcare professionals, policy makers, students, and the public about genetics and the complex issues that result from genomic research. One goal of the ELSI program is to address the implications of vastly increased genetic information and protocols on individuals and society. Another ELSI goal is to identify and develop appropriate policy options to confront and contain future ELSI problems. Because we know that “genetic information” has been misused previously in the United States and other countries, we must ensure that such mistakes are never repeated. Both the DOE and the NIH are optimistic that the ELSI program can contribute to the integration of HGP results in ways that are less disruptive, painful, or destructive than those in the past (Jin, 2000). The list of ELSI issues is long and virtually all of them have legal ramifications. They include the fair use of genetic information; the impact on genetic counseling and medical practice; the effects on personal reproductive decisions; past uses and misuses of genetic information; privacy implications of personal genetic information in various settings, e.g., the work place, schools, or in the context of adoptions; issues of the commercialization and intellectual property protection of

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genome results, including DNA sequences; conceptual and philosophical implications; implications of personal genetic variation; and genetic literacy and the understanding of genetic information, particularly information related to complex conditions that involve multiple genes and genetic-environmental interactions (Tsai and Chen, 2000). This last category, involving health issues like mental illness, heart disease, liver disease, diabetes, or cancer (Ellerin et al., 2005), represents the most complex of ELSI issues because the underlying science is poorly understood (Van Leeuwen and Bernat, 2006). For example, informing a woman that she carries an allele at BRCA1 that is associated with a high lifetime risk of developing breast cancer is a serious issue, particularly if treatment options are difficult, painful, debilitating, or oftentimes less than successful. However, other individuals or entities may want to know about such conditions, including insurers and employers (who often are responsible for insuring their employees). The extent of this protection is undoubtedly an issue that courts will have to adjudicate. We see many ELSI challenges in the future. Informed consent for participants in genetic research will remain an important issue. Genetics involves shared familial information, and the diagnosis of one person has direct implications for his or her family members. It is extremely important that patients and research participants understand what information and future predictive insights about them may emerge from genetic studies, particularly when they involve genetic testing or screening for multigenic and predisposition diseases. For example, over 600 mutations in the gene for the cystic fibrosis transmembrane regulator can lead to cystic fibrosis. Many experts think that only seven of these mutations are responsible for 85% of the cases of cystic fibrosis seen clinically, and it is these seven for which most people are tested. However, a negative test for cystic fibrosis disease-associated alleles does not necessarily mean that a person does not have a risk for cystic fibrosis. The gene for breast cancer susceptibility (BRCA1) is another case to point. Over 150 alleles in the gene have been discovered. The BRCA1 region is very large, and the number of alleles that actually predispose to breast cancer is not yet known. We also know very little about other influences that are necessary (along with one of these mutations) for breast cancer. What do you tell someone who tests positive for a disease-associated allele when you can only be vague about its clinical implications? What responsibilities do physicians and counselors have in the communication of risk information to patients when the risks themselves are poorly understood? What liability issues accompany genetic information? Can genetic information be “owned” and, if so, by whom and under what circumstances? These and other issues that arise from genetic information will challenge the courts and will be exacerbated as we get better at “reading” and interpreting the content of our genomes.

8.5 Genes and Disease The Human Genome Project is an international effort to complete the sequencing of the 100,000 genes that comprise the human genome. Upon conclusion of the Project, the genes responsible for single gene deficiency diseases, e.g., Huntington’s disease

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and cystic fibrosis, as well as for multifactorial diseases, e.g., atherosclerosis and cancer have been identified. The Project seems almost like science fiction. It is easy to think of it as not having immediate policy implications. Gene mutation and variation may result in hereditary disease, cancer, hypertension, and even susceptibility to infectious diseases. A complete compilation of all human genes (the human genome) now allow a better understanding of the role of specific genes in diseases and, consequently, better design of effective treatments. Approximately 5,000 human diseases are currently thought to have some genetic component. Some of the fundamental causes are easy to identify. In the case of Down’s syndrome for instance, there is an extra copy of chromosome 21, which is visible under the light microscope. Other genetic diseases that are strongly under the control of a single gene have also been relatively easy to identify. In the case of sickle cell anemia (SCA), a change in a single nucleotide of one gene alters a codon so that a different amino acid is incorporated into hemoglobin, the protein responsible for carrying oxygen in the blood. Such a change, which is called a single nucleotide polymorphism (SNP), may cause a change in an amino acid that can have a range of effects depending on where the amino acid is located in the protein and the change in amino acid. An SNP can also occur without changing the amino acid because many amino acids have more than one codon. In Huntington’s disease, another disease that is strongly determined by genetics, a single gene has a series of nucleotides repeated many times. In the case of SCA the gene is known as “recessive” because it requires two copies of the gene, one from each parent, to produce the disease in the offspring. If one parent donates the gene for the disease and the other does not, the child will not get the disease. In the case of Huntington’s, the gene is “dominant”; that is, if one parent has the gene, then half of his or/her sperm eggs will carry the disease and a child who receives the gene will get the disease. Thus, a child whose parent has Huntington’s has a 50% chance of getting the disease. As a result of strong single gene control over some diseases, close to 1,000 genes for a variety of diseases have been identified and localized to a chromosome through inheritance patterns. The genes involved in other diseases with some genetic component, such as cancer, heart disease and mental illness, are more difficult to identify because they involve multiple genes that are also heavily influenced by environmental factors. Environment is only one of many factors that control the degree to which harmful changes in a gene, or combination of genes, may cause health problems. Potentially harmful changes in genes may not result in problems if: • • • • • •

associated environmental factors do not turn these genes on; there is only a partial set of the combination of genes underlying a disease; the gene or genes are not expressed strongly; the change leads to only a mild form of the disease; the gene is recessive and only one copy is present; Genetic damage does not result from environmental substances or ageing.

The extent to which health problems result from potentially harmful changes to genes is therefore very difficult to predict. At one extreme, we know that diseases

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The Existence of Genetic Information

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such as Huntington’s and SCA are strongly determined by changes in a single gene. As more genes become involved and environmental factors come into play, the genetic disposition to a disease becomes less and less obvious, however, diseases that are heavily influenced by environmental factors and interaction with other genes are likely to be far more common than diseases with very strong genetic components. Many of the ELSI implications are not new. The gene for Huntington’s disease was discovered in 1993, after a 10-year search following the localization of the gene to chromosome 4 in 1983. A test for the disease was developed soon after. Many of the questions currently being addressed by the ELS issues program of the HGP have, therefore, been familiar for many years to families afflicted with Huntington’s. As a result of the HGP, however, society as a whole will have to deal much more frequently with issues arising from knowledge of the human genome. Moreover, the implications may be less clear in the case of genes identified for diseases that have strong environmental aspects and involve interaction with many other genes. Even strongly deterministic genes, such as those for Huntington’s, have a range of effects; for example, some people with the Huntington’s gene live much longer than others. Clinical issues include the education of doctors and other health service providers, patients, and the general public in genetic capabilities, scientific limitations, and social risks; and implementation of standards and quality-control measures in testing procedures. Uncertainties are associated with gene tests for susceptibilities and complex conditions (e.g., heart disease) linked to multiple genes and gene-environment interactions.

8.6 The Existence of Genetic Information The existence of genetic information with respect to individuals and the human population as a whole will have a profound impact on our day-to-day lives and may well change how we regard ourselves and one another. The knowledge of predisposition to a certain disease and the ability to design “tailor made” therapies may greatly help in the treatment of disease. Already a company in Great Britain has applied for a patent on a device that can apparently detect different forms of over 2,500 genes said to be associated with traits including behavior and intelligence. It has been argued, however, that it is not proper, particularly at this juncture in history, to search for such knowledge. For example, some have pointed out that science has often been co-opted as a tool to accentuate racial differences and to defend racist practices. Given that humans are far from resolving issues of race, it is thought that information from the HGP, and such follow-up projects as the Human Genome Diversity Project, may have the potential to inflame racism in an already overly racist world. Equally, some feel that if the goal of the HGP is to prevent disability and disease, increase life spans, decrease infant mortality, and increase intelligence, the money would be far better spent elsewhere. Given that we already know that environmental and social factors can influence such diseases as diabetes in aboriginal

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populations and drug addiction among the socially marginalized, some consider it unconscionable to dispense limited resources looking for genetic causes for these diseases. The legal aspects of knowledge of the human genome are enormous. Already DNA evidence is being used as a powerful legal tool, particularly in exonerating wrongly accused individuals. Does this mean that the criminal system should be able to keep a bank of DNA information on anyone accused and/or convicted of a crime? Could the database be used for other purposes than simply identifying and eliminating suspects? A DNA database could contain much more information on individuals, both guilty and innocent, than does the current system of taking fingerprints. On a more hypothetical note, should genes leading to a propensity for criminal activity be found, could they be used as prosecution or defense evidence in a trial? For instance, is a suspect who knows that he or she has a genetic disposition toward criminal behavior and does nothing to avoid provoking such behavior, guilty of a more serious crime than a suspect who is ignorant of having such a propensity? On the other hand, could genetic disposition be used as a defense on the grounds that the crime was really the fault of the gene, not the person? When a patient tests positive for a gene linked to risk of disease, does the physician (or the patient) have a legal responsibility to inform the patients’ relatives of their own risks? Suppose a patient finds out that she has a genetic propensity for breast cancer, but neither she nor her doctor informs her relatives; would a relative who later developed that form of cancer be able to sue, on the grounds that the genetic information had not been disclosed? Ensuring that the judge and jury in a trial are sufficiently educated to deal with these issues is yet another problem with which the legal system will have to deal. On a larger social scale, knowledge of the human genome could be used to emphasize the similarities among all humans. The genetic differences between people within an identified group have already been shown to be greater than the differences between groups. In other words, people within an “ethnic” population are more different from each other than the group as a whole is different from other “ethnic” groups. This fact is unlikely, however, to deter those who wish to emphasize any ethnic differences that may be found. On a more individual level, the results of the HGP might encourage people to view themselves as being wholly under the control of their genes. What has traditionally been viewed as the human spirit might in future be seen as limited by pre-programming at birth? Thus, though we cannot predict exactly how knowledge of the human genome will affect society, it could clearly have important consequences. Individual decisions, such as choices with respect to mates and reproduction, could also be influenced by knowledge of genetic makeup. Awareness of personal genetic differences from a perceived norm might lead to confusion and uncertainty about the potential for disease, particularly in the absence of adequate professional consultation. Genetic analysis might reveal a myriad of genetic flaws that may or may not lead to disease, depending on what they are and how they interact with the environment. How will individuals select from a debilitating array of lifestyle choices, none of which has a certain outcome? Again, analysis of one’s own genetic

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makeup could reveal the genetic makeup of parents and siblings, including, for example, unsuspected information about paternity. How willing would people be to share this knowledge and, if they decided to withhold it, how would they be affected by living with the secret?

8.6.1 Genetic Treatment of Disease From the outset, one of the defining goals of the HGP has been its potential for molecular medicine. The concept is that, once the functions of genes are known and we understand the effects of malfunctioning genes, we will be able to correct the problem either through the use of designer drugs or by replacing the faulty gene. It is the latter option that has created the most controversy. There are two routes to replacing a faulty gene. The first route, germ line therapy, has the goal of replacing a harmful gene in a fertilized human egg with a properly functioning gene that would be passed on to future generations. The other route, somatic gene therapy, aims to replace the gene in target organs or tissues of an adult, so as to fix the symptoms in that individual but not in the next generation. Germ line therapy has the more profound ethical, legal and social implications. As yet germ-line therapy in humans is not possible and some have argued that it will continue to be so for the foreseeable future. While this kind of therapy may be a long way off, it would bring, on the one hand, the hope of eradicating some genetic diseases but, on the other hand, the spectra of eugenics. The eradication of disease through germ-line therapy might not seem, by itself, to raise many ethical questions. After all, humans have eradicated the smallpox virus from the world, why not eradicate diseases with genetic components? Do doctors not have the moral obligation to provide the very best treatment to their patients and would not the eradication of the disease be more cost effective in the long run than continually treating adults with somatic gene therapy? The main ethical problem arises in defining a “treatable” disease. Some might say that eradication of a genetic disease for which there is no treatment and which is always fatal, should be pursued with all means possible. Others say that this would be the start of a slippery slope moving on toward the treatment of less obvious diseases and then to genetic enhancement. Some argue that if the technology is advanced in order to eradicate some diseases, it will inevitably be used by parents wishing to “enhance” their children, giving them the genes for raven black hair and blue eyes or athletic prowess. It was serious ethical concerns about genetic enhancement. Another ethical consideration with respect to germ-line therapy is defining what is normal, what a disability is, and what a disease is. Which of the genetic variations within a population ought to be eradicated, if any? In trying to eradicate a certain variation, are we demeaning those in the population who currently carry the gene? Somatic gene therapy has its own, less controversial, set of ELSI implications. These may be less ominous than eugenics but are of perhaps more immediate concern, given the more advanced state of the technology. Effectively, gene therapy

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involves the introduction of a properly functioning gene into target tissues in the hopes that it will be translated into a properly functioning protein, which will mask the malfunctioning protein. Often the new gene is placed into a modified virus, which is then introduced into a patient in the hope that the gene will be introduced into a tissue and properly expressed. Such types of therapy, after much research on laboratory animals, have now reached the clinical trial stage. Unfortunately, what works for a mouse does not always work for a human being. In one highly publicized case, a patient, Jesse Gelsinger, was given an injection of a virus in the hope of introducing a protein into the liver. Mouse studies showed good absorption of the gene into the liver; however, the mouse has a much higher concentration of viral receptors on its liver cells than do humans. The virus did not absorb well into the human patient and, for still unknown reasons, created a massive immune response, causing the patient to die. The original plan for the trials had been to use the virus only on children in a coma caused by the lack of the particular liver enzyme; however, ethical and safety reviews caused the researchers to change the trial direction and use adults only. Many questions are now being asked regarding the ethics and scientific judgment of those performing such clinical trials. How well are “volunteer” patients informed of the possible risks and benefits? How objective are investigators who have equity in the companies that are funding the trials? One of the risks at this stage of gene therapy is the excessive public anticipation, created in part by some researchers, with respect to future benefits. This anticipation may turn to public distrust of science, if the benefits fail to be realized and problems such as that in the Gelsinger case continue to occur. Some clinical trials have shown positive results, and so there is still hope that somatic gene therapy will become a powerful medical tool. We believe medicine can eventually find a cure for everything that ails us. This is why the HGP genetics treatments will, of course, continue their progression. To prevent unethical prejudice, discrimination, or abuse that may occur because of genetics treatments, we must explore all ethical issues and create and implement policies on genetic research and the extent in which we allow genetics treatments to change the population.

8.6.2 Genetic Testing The ELSI goals included in the HGP are composed of issues that will arise upon completion of the human DNA sequenced. The issues include integrating the new genetic information into health care and non-clinical settings, the philosophical, theological, and ethical aspects that will arise with increasing advancements, and finally how race, ethnicity and socio-economic factors will influence the development and use of genetic services. With continued advancement of the HGP, there are many benefits and arguments in favor of the HGP. The knowledge about the effects of DNA variations between individual has lead to revolutionary new ways to diagnose, treat, and possibly prevent disorders. Many new genetic disorders have been discovered and with these,

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testing is becoming increasingly available. A good example is prenatal genetic testing. An expectant mother may have genetic testing done to determine if her child has a mutation that causes a genetic disorder. Newborn screening practices have changed since breakthroughs have occurred in genetics and mapping of the human genome. Although newborn screening has been in existence since the 1960s, today’s newborn screening practices are subsumed primarily under the umbrella of genetic testing (Kenner et al., 2008). With the knowledge from the HGP, persons may have their genetic profile available to them. Their profile could be helpful in many ways. This information can help the person to build a suitable behaviour to be followed throughout their life to remain healthy. Their genetic profile may also tip the person off to have frequent check-ups if they are at risk for a certain disease. For example, if a person knows from their genetic profile that they are at risk for heart disease, they may have a modified diet and exercise program and frequent doctor’s visits to help prevent heart disease. Genetic treatment has come about from advances in genetics projects. We can improve crops by making them more resistant to insects and disease. We can make oversized and faster growing animals by inserting growth hormones in mice, pigs, sheep, and fish, thereby increasing food production. We can also increase food production by developing animals that have larger litters and lay more eggs, engineering fruits and vegetables to stay ripe longer, and developing crops and livestock that con survive harsh conditions. In addition, we can put human genes into pigs and baboons and use their organs for transplants into humans with less rejection from the human body. Genetic treatment can be used in a variety of ways to improve the human condition. In the future it is possible we may be able to correct medical disorders by going straight to their source, the genes. If a disorder is caused by a faulty or mutated gene, doctors may be able to insert new DNA into the cells. This new DNA would replace, or knockout the action of the mutation and restore the function that was damaged by the mutation. Cystic fibrosis (CF) is one of the diseases researchers are trying to cure through this type of genetic treatment. CF is a disease in which the lungs become clogged and make respiration difficult. The disease is a result of a mutation that restricts production of an enzyme that helps make the lining of the lungs. In this treatment, doctors would make copies of the corrected gene and insert it into a virus. The virus is used as a sort of transfer device. The virus would be sprayed into the nostrils of CF patients. Here the virus would reproduce by injecting the corrected gene into other cells of the patient’s body. Doctors hope the virus would reproduce enough to help the body make the needed enzyme. Although there are many advantages that supporters of the HGP can give for reasons to continue research, there are also those that argue there are many risks and disadvantages to the HGP and the use of genetic treatment. Genetic treatment may easily be twisted to support prejudice and discrimination against others. Who is to decide if a trait is good or bad? For example, a doctor has two children patients with the same problem, but she is unsure of whether to treat them both. The two children, Sam and John, are both very short for their age. Sam does not produce enough growth hormone and will never grow above five feet. Both of his parents are close to six feet tall. John, on

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the other hand, inherited his height from his parents who are both barely over five feet. A growth hormone has been produced and is available to both patients. Both sets of parents feel their child should receive the treatment to help them grow taller. They want the treatment for their child because they believe there are advantages to being tall. The doctor has to decide whether to give the treatment to only Sam, since his height is a result of a mutation, or to give it to both children. How does the doctor decide what is fair? Neither of the children is sick. Some argue that instead of changing those traits society has deemed as undesirable, we should change the idea that different is undesirable. Genetic treatment may be used in many ways to treat, cure, and maybe even prevent many diseases, but there is a concern its use may not be limited to health. With the growing knowledge involved in genetics treatment, there is an increased opportunity for people to be able to actually change how they or their children look. People may use genetic treatment to make them look younger, thinner, or even smarter. As a result, genetic treatment may also be equated with eugenics in the future. The term has a negative connotation because it implies some people or traits are better than others are.

8.6.3 Discrimination One of the problems some fear might result from knowledge of the human genome is the emergence of a whole population of socially marginalized individuals, unable to obtain a job, a family, insurance, or health care and stigmatized by the rest of society. Insurance companies already insist that those identified at risk of Huntington’s disease must take a genetic test. If the results are positive, insurance is frequently refused. Insurance companies are on record as saying that if genetic information was available, they would use it in their risk assessment. In Canada, the refusal to insure a Huntington’s patient does not have dire consequences; in general, public insurance covers many aspects of care, though the level of care varies across the country and the coverage for pharmaceuticals is less clear. In countries without a public health insurance system, however, the plight of such a non-insured person can be a nightmare. Care may be available but finding it is very difficult. As more genetic tests become available, insurance is likely to be more and more expensive for those carrying what the insurance companies deem to be risky genes. The public insurance schemes may also start to feel the pressure for such genetic testing, and be forced to make policy decisions based on the funding available and the knowledge of genetic predisposition to disease within populations. Gene therapy is at the experimental stage at this point but will certainly be very expensive when it first comes into regular use. Who will pay for it? If not public insurance, will the therapy be available only to rich people, thus creating an ever widening gap between groups in society, based on both money and genetic inheritance?

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Impact of the Human Genome Project at the Interface Between Patent and FDA Law

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Employers may also want access to genetic information. Some genes might reveal a susceptibility to environmental damage that was incompatible with a certain workplace environment. Employers might choose to screen out workers carrying that gene rather than trying to improve the environment. Individuals with genes associated with certain behavioral traits might also be excluded from the workplace. The human genome project has led to impressive scientific advances in understanding the genetic basis of disease. To date, genetic risks associated with occupational illnesses are not well understood. Recent research, however, has uncovered an allele that appears directly related to the risk of contracting chronic beryllium disease; other chromosomal abnormalities have been identified in association with cancer. Progress from the human genome project has potential implications for predicting, screening, and diagnosing occupational diseases. Ethical issues associated with the use of genetic testing in the workplace will present employers, insurers, and physicians with challenging decisions related to promoting health in the workplace while avoiding potential misuse of sensitive genetic information.

8.7 Impact of the Human Genome Project at the Interface Between Patent and FDA Law The debate over biotechnology policy is at heart a debate over information policy. At one level, the debate covers how to provide intellectual property protection to the tools of biotechnology and the valuable information they produce, such as gene sequences and chromosome maps. At a different level, the debate shifts to questions of how best to distribute information to empower others and to prevent information misuses and loss of privacy. Aided by the new tools of the computer age, biotechnology is developing faster than any previous technology. In the process of development, biotechnology is creating a wider gap between practice and policy. Our growing ability to transform genetic information into new products and organisms is intended to enhance agriculture, fight pollution, and alter hereditary diseases which make biotechnology a powerful and threatening tool. Development of genome technology will outpace needed public policy which will be required to cope with consequences. No matter how safe and well controlled technology may be, public opinion will be driven by lack of knowledge, lack of trust of science and government and a tendency to believe that things are worse than what the public is told. Therefore, the question is: How does society ensure that its governmental and social institutions manage the future course of evolution with enough wisdom to avoid catastrophic mistakes? Two kinds of currently available genomic patents may significantly interfere with medical research: (1) patents such as those on specific single nucleotide polymorphisms (SNPs), which may include claims that control the inference of phenotypic characteristics from specific genotypes, and (2) patents on computer-based genomic

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information, databases, or manipulation procedures. These will create more serious encumbrances than will patents on expressed sequence tags (ESTs). Two approaches should be considered vis-à-vis these genomic patents: (1) Reconsideration and redefinition of the recent extensions of patentable subject matter into more and more intangible areas. This could be pursued by legislation or by test litigation to seek Supreme Court reversal of certain of the decisions of the Court of Appeals for the Federal Circuit (CAFC). (2) A narrow legislative exemption protecting the ability to use SNPs and phenotypic-genotypic relationships in medical research, including contexts in which medical research and clinical practice are substantially intertwined (Barton, 2002). Patents and licenses on gene-based diagnostic tests are properly enforceable and do not permit academic or other clinical practitioners to practice such tests without license or other authorization from the patent holder (Goldstein and Golod, 2002). The real risk of genome technology is not whether appropriate clinical trials can be designed, nor how or where inserted DNA integrates into the host cell or any other such events. The real risk may be illustrated by recalling the incident which led to the Asylomar Conference and creation of the NIH’s Guidelines for Research Involving Recombinant DNA Molecules (Guidelines) viz., The potential benefits and harms of DNA patenting for biomedical research and medical practice. It argues that, all things considered, the benefits of patenting DNA outweigh the harms, although societies should adopt policies designed to prevent or mitigate the harms associated with patenting. Some of these policies include: (1) (2) (3) (4) (5) (6)

Reinforcing the research exemption for academic researchers, Raising the “bar” for the criteria of patentability, Restricting the scope of patents, Disclosing conflicts of interest related to DNA patents, Sharing the economic benefits of patenting with patients, and Providing insurance coverage for some types of genetic tests (Resnick, 2003).

The first patent for a genetically modified organism (GMO) issued in 1988 on a transgenic mouse modified to be useful in studying cancer. Patent and FDA law may be said to “interface” in a couple of different ways. Legislative enactments such as the Orphan Drug Act 9 and the Patent Term Restoration Act10 “interface” in the sense that each modifies patent law and FDA law. However, a different kind of interface may be found in the tension between the differing policies which underlie patent and FDA law. The patent system is intended to foster technological innovation and economic progress. FDA law serves different policy objectives because it is intended to protect the public’s safety and welfare. To speak of the interface between patent law and FDA law is to speak of balancing competing policy considerations.

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There is little reason to believe that differences in opinion about the appropriateness of human gene therapy will resolve spontaneously, or even after extensive public discussion. Where there is no agreement on what decision to make, the only alternative is a process for making the decision. Government agencies must demonstrate that the process is rational and fair. Whatever process is adopted must involve professionals from many disciplines, must be open to the public and must be insulated from political pressures. The best hope of setting policy to deal with the implications of genome technology is to use an effective process for gathering necessary information and subjecting that information to appropriate and thorough consideration to formulate recommendations for appropriate bodies. The biotechnology industry can and should do better. It ought to be at the forefront of fostering a rational discussion of genome technology and its implications for social policy. If such a discussion does not begin soon, events will overtake the opportunity. Announcements of experiments made too soon, whether successful or unfortunate, will catch the public’s attention with the potentially destructive consequences of fear, mistrust and backlash.

8.8 Regulatory Process Regulation of gene transfer therapies by the Food and Development Authority (FDA) has been an exercise of fitting the existing system to the new technology. The FDA’s regulatory approach may be modified in light of additional knowledge about risks and benefits. The products for somatic-cell and gene therapy are more prudent than waiting until the field has matured. The Recombinant DNA Advisory Committee (RAC) reviews all proposals for National Institute of Health (NIH) funded research projects pursuant to the Guidelines. The Guidelines were intended to monitor biotechnology research until more was known about the safety of the organisms produced through genetic engineering. These Guidelines have been revised over the years in a manner characterized as demonstrating the gradual rise of technology to prominence over policy development to the point that the RAC has virtually relaxed itself out of a job. The RAC responded to the recommendations of the President’s Commission in its 1982 report, Splicing Life, by establishing the Human Gene Therapy Subcommittee to review proposals involving the use of rDNA techniques on human beings and to continue to explore issues that would be posed by the extension of these techniques into genetic enhancement and germline gene therapy. Thus for the short term, technologies such as gene therapy will be dealt with as drugs or biologics in the same manner as other therapies, although approval to test gene therapies in patients has been more rigorous than any other medical area. A synthetic polynucleotide sequence intended to alter a genetic sequence in human somatic cells after administration to the patient is classified as a drug, so that a new drug application (NDA) is required. A retroviral vector containing a gene to be

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administered intravenously into the patient will be classified as a biologic, requiring a product license application (PLA) and ELA (establishment license application). A retroviral vector containing a gene and intended to modify cells ex vivo is considered a biologic intended for further manufacture; hence, both a PLA and ELA will be required. Issues raised by biotechnology in the 1980s were trivial compared to those to be raised by genome technology. To resolve those issues effectively and fairly, we must develop a coherent moral and ethical basis for decisions in several areas, including patent and FDA law (Kessler et al., 1993).

8.9 Conclusion Searching for medically useful, and therefore potentially profitable, genes also raises many ethical questions. Heritable disease patterns sometimes emerge in populations that have not mixed extensively with other populations. How to regulate the gene hunters without scaring off investment is a familiar problem to governments that already have experience with charging royalties and regulating natural resource operations. Gene “mining” companies, however, present a much more complex and emotional set of ethical issues than does the natural resources sector. Many of the ethical, legal and social issues that are being discussed with respect to the Human Genome Project are not new. Genetic tests for a variety of diseases are currently available and some people are already struggling with the ethical and practical implications. What will change over the next few years, as a result of the Human Genome Project, is the scale of the issues and how society will have to cope with the greyer areas of genetic disease and disability. Dealing with a single gene that causes death or chronic disability is one issue; dealing with whole sets of genes whose impacts vary depending on environmental interactions is another. The rate of scientific advancement has tended to outstrip the legislative capacity of governing bodies and there has been some media “over hype” with respect to genetic research and its potential for treatment of disease. It will be years before many of the genetic tests are available and before genetic diseases can be treated. Society as a whole must use this time to discuss and decide on how genetic information ought to be used, before the choices are made for them. It is a discussion that those with genetic dispositions to diseases such as Huntington’s have long wanted to make more public. Understanding the ethical and moral implications of genetic information and technology is crucial towards ensuring the proper use of genetic data. The ELSI programs have had a positive influence in understanding problematic areas surrounding the HGP by acting as a center for discussion for many bioethicists and scientists. However, ELSI is often too passive and does not provide enough practical guidance to the public on the complex implications of the HGP. As it stands now, there is room for major improvement such as increased cooperation between ELSI and nongovernment organizations. NGO’s would serve as bridges between ELSI and the

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public, and collaboration would enable in-depth probing and more comprehensive analysis of issues of public concern. New knowledge in the area generated by HGP can result in new ways to diagnose, treat, and prevent many genetic disorders. Apart from medical applications, knowing about our DNA can affect the world’s condition. It can lead to problem solving in energy sources and environmental cleanup plus better health care. Learning about DNA and gaining an understanding of human biology will give us a better world and a better life, as long as we keep it within the boundaries of ethical, legal, and social concern.

References Barton, J.H., 2002, Patents, genomics, research and diagnostics. Acad Med 77:1348–1367. Durfy, S.J., 1993, Ethics and the Human Genome Project. Arch Pathol Lab Med 117(5):466–469. Ellerin, B.E., Schneider, R.J., Stern, A., Toniolo, P.G., Formenti, S.C., 2005, Ethical, legal, and social issues related to genomics and cancer research: the impending crisis. J Am Coll Radiol 2(11):919–926. Goldstein, J.A., Golod, E., 2002, Human gene patents. Acad Med 77(12Pt 2):1315–1328. Gutiérrez-Samperio, C., 2002, Bioethics in genetic engineering. Gac Med Mex 138(1):109–119. Jin, J., 2000, An evaluation of the ethical, legal and social implications program of the U.S. Human Genome Project. Princet J Bioeth Spring 3(1):35–50. Kenner, C., Lewis, J.A., Pressler, J.L., Little, C.M., 2008, Neonatal genetic testing is more than screening. Crit Care Nurs Clin North Am 20(2):233–237. Kessler, D.A., Siegel, J.P., Noguchi, P.D., Zoon, K.C., Feiden, K.L., Woodcock, J., 1993, Regulation of somatic-cell therapy and gene therapy by the food and drug administration. New Eng J Med 329(16):1169–1173. Masui, T., Takada, Y., 2003, Ethical, legal, and social issues of genome research – new phase of genome research desperately requires social understanding and safeguards on the use of medical records and other personal information. Yakugaku Zasshi 123(3):107–119. McCunney, R.J., 2002, Genetic testing: ethical implications in the workplace. Occup Med 17(4):665–672. Meslin, E.M., Thomson, E.J., Boyer, J.T., 1997, The Ethical, Legal, and Social Implications Research Program at the National Human Genome Research Institute. Kennedy Inst Ethics J 7(3):291–298. Murdoch, C., Caulfield, T., 2009, Commercialization, patenting and genomics: researcher perspectives. Genome Med 1(2):22. Resnik, D.B., 2003, Are DNA patents bad for medicine? Health policy 65(2):181–197. Tsai, S.F., Chen, P.J., 2000, Impact of human genome research on medicine – the initial Taiwan experience. J Formos Med Assoc 99(2):107–115. Van Leeuwen, D.J., Bernat, J.L., 2006, Ethical, social and legal implications of genetic testing in liver disease. Hepatology 43(6):1195–1201 Zneimer, S.M., 2002, The human genome project: exploring its progress and successes and the ethical, legal, and social implications. Clin Leadersh Manag Rev 16(3):151–157.

Chapter 9

Polymerase Chain Reaction

Abstract Kary Mullis devised a method of replicating genes called "PCR" (polymerase chain reaction). A DNA sequence less than one part in a million of the total sample can be cloned. In fact, a single gene can be amplified into millions of duplicate copies. In order to determine the exact DNA sequence of a gene or section of DNA, it is necessary to have an adequate sample of the particular gene to work with. PCR allows a researcher to replicate a gene into a workable amount. PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR has become commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. Keywords Amplification · DNA sequencing · Site specific mutation · Polymerisation · PCR · Taq polymerase · Thermus auquaticus · Polymerase chain reaction · Primer design · GC-content · Melting temperature · Tm · Template · Real-time PCR · BRCA1 · Cystic fibrosis · RT-PCR

9.1 Prologue On 10th of December 1993, Kary Mullis received the Noble prize in Chemistry from King Carl XVI Gustaf of Sweden for his invention of polymerase chain reaction. Mullis had published and patented his work in 1985. The applications of this method have revolutionised both basic and applied sciences. The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA. The development of this technique resulted in an explosion of new techniques in molecular biology as more and more applications of the method were published. The technique was made possible by the discovery of Taq polymerase obtained from Thermus auquaticus. This DNA polymerase is stable at the high M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_9,

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temperatures needed to perform the amplification, whereas other DNA polymerases become denatured. Mullis defined PCR not as a specific technique but rather as a concept. Since this technique involves amplification of DNA, the most obvious application of the method is in the detection of minuscule amounts of specific DNA. This is important in the detection of low level bacterial infections or rapid changes in transcription at the single cell level, as well as the detection of a specific individual’s DNA. It can also be used in DNA sequencing, screening for genetic disorders, site specific mutation of DNA, or cloning or subcloning of cDNAs.

9.2 The Concept of Polymerase Chain Reaction PCR, like DNA sequencing, is based on the DNA polymerization reaction. PCR is used in genome sequencing, including the Human genome project. Using the randomn primers, the entire sequence of a genome can be amplified in pieces. A primer and dNTPs are added along with a DNA template and the DNA polymerase (in this case, Taq). The main concern with PCR is that, in addition to using a primer that sits on the 5 end of the gene and makes a new strand in that direction, a primer is made to the opposite strand to go in the other direction. The original template is melted (at 94◦ C), the primers anneal (@ 45–55◦ C) and the polymerase makes two new strands (@ 72◦ C), doubling the amount of DNA present. This provides 2 new templates for the next cycle. The DNA is again melted, primers anneal, and the Taq makes 4 new strands. A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. The elongation temperature depends on the DNA Polymerase. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. Prior to the use of thermostable DNA polymerases in PCR, researchers had to laboriously replenish the reaction with fresh enzyme (such as Klenow or T4 DNA polymerase) after each denaturation cycle. Thermostable DNA polymerases revolutionized and popularized PCR because of their ability to withstand the high denaturation temperatures. The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of primer annealing. Thermostable DNA polymerases can also be used for either one-enzyme or two-enzyme RT-PCR (Myers and Gelfand, 1991; Chiocchia and Smith, 1997). For example, Tth DNA polymerase can act as a reverse transcriptase in the presence of Mn2+ for one-enzyme RT-PCR (Myers and Gelfand, 1991). All of the DNA polymerases mentioned can be used for amplification of the first-strand cDNA produced by a reverse transcriptase, such as AMV RT, in two-enzyme RT-PCR. The thermostable DNA polymerases can be divided into two groups, those with a 3 →5 exonuclease (proofreading) activity, such as Pfu DNA polymerase, and those without the proofreading function, such as Taq DNA polymerase. These two groups have some important differences. Proofreading DNA polymerases are more accurate than nonproofreading polymerases due to the 3 →5 exonuclease activity, which

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The Concept of Polymerase Chain Reaction

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Fig. 9.1 Concept of PCR

can remove a misincorporated nucleotide from a growing chain of DNA. When the amplified product is to be cloned, expressed or used in mutation analysis, Pfu DNA polymerase is a much better choice due to its high fidelity. However, for routine PCR, where simple detection of an amplification product is the goal, Taq DNA polymerase is the most commonly used enzyme because yields tend to be higher with a nonproofreading DNA polymerase (Fig. 9.1). Taq DNA polymerase (Taq) can amplify long DNA fragments, if the conditions were set properly. Taq could successfully perform the “long PCR” up to 24 kb. The conditions include: (1) longer primers, (2) a 2-step cycling, and (3) a “long buffer.” The most important requirements are the survival rate of Taq at high temperatures and that of the primers against the 5 –3 exonuclease activity of Taq (Lee et al., 2009). Amplification with nonproofreading DNA polymerases results in the templateindependent addition of a single nucleotide to the 3 -end of the PCR product, whereas the use of proofreading DNA polymerases results in blunt-ended PCR products (Hu, 1993). The single-nucleotide overhang can simplify the cloning of PCR products. Proofreading DNA polymerases are also used in blends with nonproofreading DNA polymerases, or amino-terminally truncated versions of Taq DNA polymerase, to amplify longer stretches of DNA with greater accuracy than the nonproofreading DNA polymerase alone (Cline et al., 1996). A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations (errors) in the DNA sequence, since it lacks 3 →5 proofreading exonuclease activity. Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied DNA sequence. However these enzymes polymerize DNA at a much slower rate than Taq.

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Combinations of both Taq and Pfu are available nowadays that provide both high processivity (fast polymerization) and high fidelity (accurate duplication of DNA). Polymerase chain reaction (PCR) is widely used in different areas of science and biotechnology. Grouping of PCR primer sets for amplification in a single tube (multiplexing) provides substantial savings in terms of time, chemicals and, most importantly sample materials. Thus, multiplexing is a powerful way for optimizing the cost of genetic analysis and some procedures of PCR are patented (Piepenbrock et al., 2004), which further demonstrates its importance. While a lot of effort has been put into the experimental optimization of multiplex PCR conditions (Henegariu et al., 1997; Zangenberg et al., 1999), little attention has been paid to the prediction of primer compatibility in multiplex reactions. Smaller multiplex groups are often composed manually and verified by trial and error, but this kind of approach is suboptimal for large datasets. Although there are software programs available for testing the compatibility of PCR primer pairs for multiplexing (Rychlik, 1995; Vallone and Butler, 2004), these do not perform automatic grouping and are unable to handle large datasets automatically.

9.3 PCR Optimisation The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying small quantities of DNA. Each step of the cycle should be optimized for each template and primer pair combination. Successful amplification depends on many factors viz. DNA template quantity and quality, magnesium ions as cofactors of Taq polymerase etc.

9.3.1 Magnesium Concentration Magnesium is a required cofactor for thermostable DNA polymerases, and magnesium concentration is a crucial factor that can affect the success of the amplification. Template DNA concentration, chelating agents present in the sample (e.g., EDTA or citrate), dNTP concentration and the presence of proteins can all affect the amount of free magnesium in the reaction. Excess free magnesium reduces enzyme fidelity (Eckert and Kunkel, 1990) and may increase the level of nonspecific amplification (Williams, 1989; Ellsworth et al., 1993).

9.3.2 Buffer Concentration Most reaction buffers consist of a buffering agent, most often a Tris-based buffer, and salt, commonly KCl. The buffer regulates the pH of the reaction, which affects the DNA polymerase activity and fidelity. Modest concentrations of KCl will increase DNA polymerase activity by 50–60% over activities in the absence of KCl; 50 mM KCl is considered optimal (Gelfand and White, 1990).

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9.3.3 Enzyme Concentration 1–1.25 units of Taq DNA polymerase in a 50 μl amplification reaction is usually recommended. In most cases, this is an excess of enzyme, and adding more enzyme will not significantly increase product yield. In fact, increased amounts of enzyme increase the likelihood of generating artifacts associated with the intrinsic 5 →3 exonuclease activity of Taq DNA polymerase, resulting in smeared bands in an agarose gel (Longley et al., 1990; Bell and DeMarini, 1991).

9.3.4 PCR Primer Design PCR primers define the target region to be amplified and generally range in length from 15–30 bases. Ideally primers will have a GC-content of 40–60%. Avoid three G or C residues in a row near the 3 -end of the primer to minimize nonspecific primer annealing. Also, avoid primers with intra- or intermolecular complementary sequences to minimize the production of primer-dimer. Intramolecular regions of secondary structure can interfere with primer annealing to the template and should be avoided. Ideally, the melting temperature (Tm ), the temperature at which 50% of the primer molecules are annealed to the complementary sequence, of the two primers will be within 5◦ C, so the primers anneal efficiently at the same temperature. Primers can be designed to include sequences that can be useful for downstream applications. For example, restriction enzyme sites can be placed at the 5 -ends of the PCR primers to facilitate subsequent cloning of the PCR product, or a T7 RNA polymerase promoter can be added to allow in vitro transcription without the need to subclone the PCR product into a vector.

9.3.5 Template Quality Successful amplification depends on DNA template quantity and quality. Reagents commonly used in the purification of nucleic acids (salts, guanidine, proteases, organic solvents and SDS) are potent inactivators of DNA polymerases. For example, 0.01% SDS will inhibit Taq DNA polymerase by 90%, while 0.1% SDS will inhibit Taq DNA polymerase by 99.9% (Konat et al., 1994). A few other examples of PCR inhibitors are phenol (Katcher and Schwartz, 1994), heparin (Beutler et al., 1990; Holodniy et al.,1991), xylene cyanol, bromophenol blue (Hoppe et al., 1992), plant polysaccharides (Demeke and Adams, 1992), and the polyamines spermine and spermidine (Ahokas and Erkkilä, 1993). In some cases, the inhibitor is not introduced into the reaction with the nucleic acid template. If an amplification reaction fails and you suspect the DNA template is contaminated with an inhibitor, add a control DNA and primer pair that has amplified well in the past to the amplification reaction with the suspect DNA preparation. Failure to amplify the control DNA usually indicates the presence of an inhibitor. Additional

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steps to clean up the DNA preparation, such as phenol/chloroform extraction or ethanol precipitation, may be necessary.

9.3.6 Template Quantity The amount of template required for successful amplification depends upon the complexity of the DNA sample. Common mistakes include using too much plasmid DNA, too much PCR product or too little genomic DNA as the template. Reactions with too little DNA template will have low yields, while reactions with too much DNA template can be plagued by nonspecific amplification.

9.3.7 Cycling Parameter The two most commonly altered cycling parameters are annealing temperature and extension time. The lengths and temperatures for the other steps of a PCR cycle do not usually vary significantly. However in some cases, the denaturation cycle can be shortened or a lower denaturation temperature used to reduce the number of depurination events, which can lead to mutations in the PCR products. Primer sequence is a major factor that determines the optimal annealing temperature, which is often within 5◦ C of the melting temperature (Tm ) of the primers. Using an annealing temperature slightly higher than the primer Tm will increase annealing stringency and can minimize nonspecific primer annealing, decreasing the amount of undesired products synthesized. However, using an annealing temperature lower than the primer Tm can result in higher yields, as the primers anneal more efficiently at the lower temperature. Numerous formulas exist to determine the theoretical Tm of nucleic acids (Baldino, Jr. et al., 1989; Rychlik et al., 1990). The formula below can be used to estimate the melting temperature for oligonucleotides: Tm = 81.5 + 16.6 × (log10 [Na+ ]) + 0.41 × (%G+C) – 675/n where [Na+ ] is the molar salt concentration, [K+ ] = [Na+ ] and n = number of bases in the oligonucleotide. The length of the extension cycle, which may also need to be optimized, depends on the size of the PCR product and the DNA polymerase being used. In general, allow approximately 1 min for every 1 kb of amplicon (minimum extension time = 1 min) for nonproofreading DNA polymerases and 2 min for every 1 kb of amplicon for proofreading DNA polymerases. Avoid excessively long extension times, as they can increase the likelihood of generating artifacts associated with the intrinsic 5 →3 exonuclease activity of Taq DNA polymerase (Longley et al., 1990; Bell and DeMarini, 1991). PCR typically involves 25–35 cycles of amplification. The risk of undesirable PCR products appearing in the reaction increases as the number of cycles increases, so we recommend performing only enough cycles to synthesize the desired amount of product. If nonspecific amplification products accumulate before sufficient amounts of PCR product can be synthesized, consider diluting the products of the first reaction and performing a second amplification with the same

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primers or primers that anneal to sequences within the desired PCR product (nested primers).

9.3.8 PCR Enhancers and Additives The addition of PCR-enhancing agents can increase yield of the desired PCR product or decrease the production of undesired products. There are many PCR enhancers, which can act through a number of different mechanisms. These reagents will not enhance all PCR reactions; the beneficial effects are often template- and primer-specific and will need to be determined empirically. Some of the more common enhancing agents are discussed below. The addition of betaine, DMSO and formamide can be helpful when amplifying GC-rich templates and templates that form strong secondary structures, which can cause DNA polymerases to stall. GC-rich templates can be problematic due to inefficient separation of the two strands of DNA or the tendency for the complementary, GC-rich primers to form intermolecular secondary structures, which will compete with primer annealing to the template. Betaine reduces the amount of energy required to separate the strands of DNA templates (Rees et al., 1993). DMSO and formamide are thought to aid in amplification in a similar manner by interfering with the formation of hydrogen bonds between the two strands of DNA (Geiduschek and Herskovitzs, 1961). Some reactions that amplify poorly in the absence of enhancers will give a higher yield of PCR product when betaine (1 M), DMSO (1–10%) or formamide (1–10%) are added. Concentrations of DMSO greater than 10% and formamide greater than 5% can inhibit Taq DNA polymerase and presumably other DNA polymerases as well (Varadaraj and Skinner, 1994). Specific examples of the effects of DMSO and betaine have been published (Frackman et al., 1998). In some cases, general stabilizing agents such as BSA (0.1 mg/ml), gelatin (0.1– 1.0%) and nonionic detergents (0–0.5%) can overcome failures to amplify a region of DNA. These additives can increase DNA polymerase stability and reduce the loss of reagents through adsorption to the tube walls. BSA has also been shown to overcome the inhibitory effects of melanin on RT-PCR (Giambernardi et al., 1998). R R X-100, have the NP-40, and Triton Nonionic detergents, such as Tween-20, added benefit of overcoming the inhibitory effects of trace amounts of strong ionic detergents, such as 0.01% SDS (Gelfand and White, 1990). Ammonium ions can make an amplification reaction more tolerant of nonoptimal conditions. For this reason, some PCR reagents include 10–20 mM (NH4 )2 SO4 . Other PCR enhancers include glycerol (5–20%), polyethylene glycol (5–15%) and tetramethyl ammonium chloride (60 mM).

9.3.9 Nucleic Acid Cross-Contamination It is important to minimize cross-contamination between samples and prevent carryover of RNA and DNA from one experiment to the next. Use separate work areas

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and pipettors for pre- and post-amplification steps. Use positive displacement pipets or aerosol-resistant tips to reduce cross-contamination during pipetting. Wear gloves and change them often. There are a number of techniques that can be used to prevent the amplification of DNA contaminants. PCR reagents can be treated with isopsoralen, a photo-activated, cross-linking reagent that intercalates into double-stranded DNA molecules and forms covalent, interstrand crosslinks, to prevent DNA denaturation and replication. These interstrand crosslinks effectively render contaminating DNA unamplifiable. Treatment of the PCR reagents with uracil-N-glycosylase (UNG), a DNA repair enzyme that hydrolyzes the base-ribose bond at uracil residues, eliminates one of the most common sources of DNA contamination: previously amplified PCR products. UNG treatment prevents replication of uracil-containing DNA by causing the DNA polymerase to stall at the resulting abasic sites. For UNG to be an effective safeguard against contamination, the products of previous amplifications must have been synthesized in the presence of dUTP. This is easily accomplished by substituting dUTP for some or all of the dTTP in the reaction. Nonproofreading polymerases will readily incorporate dUTP into a PCR product, but proofreading polymerases incorporate dUTP much less efficiently (Slupphaug et al., 1993; Greagg et al., 1999; Lasken et al., 1996). Since the incorporation of dUTP has no noticeable effect on the intensity of ethidium bromide staining or on the electrophoretic mobility of the PCR product, the reactions can be analyzed by standard agarose gel electrophoresis. While both methods are effective (Rys and Persing, 1993), UNG treatment has the advantage that both single-stranded and double-stranded DNA templates will be rendered unamplifiable (Longo et al., 1990).

9.4 Advances in the PCR Technique The advent of the polymerase chain reaction (Hooberman, 1992; Chan and Greiner, 1994) has provided an alternative to Southern blot analysis and has the following advantages: it is technically simpler and has a much faster turnaround time; the amount of clinical materials required is much smaller; and in most situations, the test can be performed on archival, formalin-fixed, paraffin-embedded materials. Modifications of the technique to include reverse transcription make it possible to use RNA as the starting material (reverse transcription polymerase chain reaction; Elmberger et al., 1995). With PCR, it is routinely possible to amplify enough DNA from a single hair follicle for DNA typing. Some workers have successfully amplified DNA from a single sperm cell. The PCR technique has even made it possible to analyze DNA from microscope slides of tissue preserved years before. However, the great sensitivity of PCR makes contamination by extraneous DNA a constant problem. The polymerase chain reaction is a technique for quickly “cloning” a particular piece of DNA in the test tube (rather than in living cells like E. coli). PCR is used also used to detect mutations after sequencing. Recently DNA genoR probes has been reported in detection of typing with mutation specific TaqMan

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single nucleotide polymorphism (Li, 2009) using coamplification at low denaturation temperature – PCR (COLD-PCR) .This helped to identify low level mutation in clinical samples and to identify tumor protein p53 (TP 53) and epidermal growth factor receptor (EGFR) mutations in tumors. Recent technical advances have allowed the accurate quantitation of the template in real time (Erlich et al., 1991; Gerrard et al., 1998; Orlando et al., 1998; Luthra et al., 1998). The latter has major implications in the monitoring of residual disease.

9.4.1 RT-PCR The discovery of reverse transcriptases, or RNA-dependent DNA polymerases, and their role in retrovirus infection (Baltimore, 1970; Temin and Mizutani, 1970) altered molecular biology’s central dogma of DNA→RNA→protein. Reverse transcriptases use an RNA template to synthesize DNA and require a primer for synthesis, like other DNA polymerases. RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RTPCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.

9.4.2 “Hot Start” PCR Hot Start PCR allows the inhibition of polymerase activity during PCR reaction preparation (Birch, 1996). By limiting polymerase activity prior to PCR cycling, Hot Start PCR reduces non-specific amplification and increases PCR product target yield. To perform hot-start PCR, the reactions are assembled on ice or at room temperature, but one critical component is omitted until the reaction has been heated to 60–65◦ C, at which point the missing reagent is added. This omission prevents the polymerase from extending primers until the critical component is added at the higher temperature where primer annealing is more stringent. Hot Start PCR is commonly performed by using included chemical modifications (Kainz et al., 2000), wax-barrier methods (Horton et al., 1994, Bassam and Caetano-Anolles, 1993) and inhibition by a Taq-directed antibody (Takahashi et al.,1996). However, this method is tedious and increases the risk of contamination. A second, less labor-intensive approach involves the reversible inactivation or physical separation of one or more critical components in the reaction. For example, the magnesium or DNA polymerase can be sequestered in a wax bead, which melts as the reaction is heated to 94◦ C during the denaturation step, releasing the component only at higher temperatures (Krishnan et al., 1991). Alternatively, the DNA polymerase can be kept in an inactive state by binding to an oligonucleotide, also known as an aptamer, (Lin and Jayasena, 1997) or an antibody (Scalice et al., 1994).

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This bond is disrupted at the higher temperatures, releasing the functional DNA polymerase.

9.4.3 Long Range PCR Long range PCR allows the amplification of PCR products, which are much larger than those achieved with conventional Taq polymerases. Up to 27 kb fragments are possible from good quality genomic DNA, although 10–20 kb fragments are routinely achievable, given the appropriate conditions. The method relies on a mixture of thermostable DNA polymerases, usually Taq DNA polymerase for high processivity (i.e. 5 -3 polymerase activity) and another DNA polymerase with 3’-5’ proofreading abilities (usually Pwo). This combination of features allows longer primer extension than can be achieved with Taq alone. Amplification of long DNA fragments is desirable for numerous applications such as physical mapping applications (Rose, 1991) and direct cloning from genomes.

9.4.4 Inverse PCR Inverse PCR is one of numerous tools utilized for furthering our understanding of the complexities of DNA, and facilitates cloning and sequencing of otherwise unknown elements of a genome. Inverse PCR (IPCR), described by Ochman et al. in 1988, is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle. Inverse PCR has many applications in molecular genetics, for example, the amplification and identification of sequences flanking transposable elements. Mostly it is done when bordered sequences of genes are not known. The borders of the vectors are used as primers and thus polymerisation proceeds in inverse direction i.e. away from the vector sequence flanked by the primers towards the DNA sequence of inserted segment.

9.4.5 Anchored PCR This is applicable when only one of the two ends of the DNA sequence is to be amplified. In such cases only one primer instead of two primers are used .Variety of polymerase chain reaction in which only enough information is known to make a single primer. A known sequence is thus added to the end of the DNA, perhaps by enzymic addition of a polynucleotide stretch or by ligation of a known piece of DNA. The PCR can then be performed with the gene-specific primer and the anchor primer.

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9.4.6 Nested Primer PCR PCR amplification is performed with one set of primers and then some product is taken with or without removal of reagents for re-amplification with an internallysituated, “nested”set of primers. This process adds another level of specificity, meaning that all products non-specifically amplified in the first round will not be amplified in the second. Nested PCR uses two sets of amplification primers (Gong et al., 2002). The target DNA sequence of one set of primers (termed “inner” primers) is located within the target sequence of the second set of primers (termed “outer” primers). In practice, a standard PCR reaction is first run with the patient sample using the “outer primers”. Then a second PCR reaction is run with the “inner primers” using the product of the first reaction as the amplification target. This procedure increases the sensitivity of the assay by reamplifying the product of the first reaction in a second reaction. The specificity of the assay is increased because the inner primers amplify only if the first PCR reaction yielded a specific product.

9.4.7 Colony PCR Colony PCR is the screening of bacterial (E. coli) or yeast clones for correct ligation or plasmid products (Plourde-Owobi et al., 2005). Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest.

9.4.8 Quantitative PCR PCR and RT-PCR are generally used in a qualitative format for evaluating biological samples. However, a wide variety of applications, such as the determining viral load, measuring responses to therapeutic agents and characterizing gene expression, would be improved by quantitative determination of target abundance. Theoretically, this should be easy to achieve, given the exponential nature of PCR, because a linear relationship exists between the number of amplification cycles and the logarithm of the number of molecules. In practice however, the actual efficiency of amplification is decreased because of contaminants (inhibitors), competitive reactions, substrate exhaustion, inactivation of the polymerase and target reannealing. As the number of cycles increases, the amplification efficiency decreases, eventually resulting in a plateau effect. Normally, quantitative PCR requires the measurement to be taken before the plateau phase, so the relationship between the number of cycles and molecules is relatively linear. This point must be determined empirically for different reactions because of the numerous factors that can affect the amplification efficiency. Because the measurement is taken prior to the reaction plateau, quantitative PCR uses fewer

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amplification cycles than basic PCR. This can cause problems in the detection of the final product because there is less product to detect. To monitor the efficiency of amplification, many applications are designed to include an internal standard in the PCR. One such approach includes a second primer pair that is specific for a “housekeeping” gene (i.e., a gene that has constant expression levels among the samples compared) in the reaction (Gaudette and Crain, 1991). Amplification of housekeeping genes verifies that the target nucleic acid and reaction components were of acceptable quality but does not account for differences in amplification efficiencies due to differences in product size or primer annealing efficiency between the internal standard and target being quantified. The concept of competitive PCR, a variation of quantitative PCR is a response to this limitation. In competitive PCR, a known amount of a control template is added to the reaction. This template is amplified using the same primer pair as the experimental target molecule but yields a distinguishable product (e.g., different size, restriction digest pattern, etc.). The amounts of control and test product are compared after amplification. While these approaches control for the quality of the target nucleic acid, buffer components and primer annealing efficiencies, they have their own limitations (Siebert and Larrick, 1993; McCulloch et al., 1995), including the fact that many depend on final analysis by electrophoresis. Numerous fluorescent solution and solid-phase assays have been described to measure the amount of amplification product generated in each reaction, but they can fail to discriminate amplified DNA of interest from nonspecific amplification products. Some of these analyses rely on blotting techniques, which introduces another variable due to nucleic acid transfer efficiencies, while other assays have been developed to eliminate the need for gel electrophoresis yet provide the requisite specificity. Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way of overcoming the need for analysis by electrophoresis.

9.4.9 Real-Time PCR The use of fluorescently labeled oligonucleotide probes or DNA-binding fluorescent R green, to detect and quantitate a PCR product allows quandyes, such as SYBR titative PCR to be performed in real time (Antwerpen, 2008). DNA-binding dyes are easy to use but do not differentiate between specific and nonspecific PCR products. Fluorescently labeled nucleic acid probes have the advantage that they react with only specific PCR products. These probes can also be used to detect single nucleotide polymorphisms (Bernard et al., 1998). Real-time PCR using labeled oligonucleotide probes employs two different fluorescent reporters and relies on the transfer of energy from one reporter (the energy donor) to the second reporter (the energy acceptor) when the reporters are in close proximity. The second reporter can be a quencher or a fluor. If the second reporter is a quencher, the energy from the first reporter is absorbed but re-emitted as heat rather than light, leading to a decrease in the fluorescent signal. Alternatively, if

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the second reporter is a fluor, the energy can be absorbed and re-emitted at another wavelength through fluorescent resonance energy transfer (FRET), and the progress of the reaction can be monitored by the decrease in fluorescence of the energy donor or the increase in fluorescence of the energy acceptor. During the exponential phase of PCR, the change in fluorescence is proportional to the accumulation of PCR product. To simplify quantitation, specially designed instruments perform both the thermal cycling steps to amplify the target and the fluorescence detection to monitor the change in fluorescence in real time during each PCR cycle. Real time reverse PCR is a modification of the RT-PCR and is different from other quantitative PCR as it quantitates the initial amount of the template instead of detecting the amount of final amplified product.

9.4.10 Rapid Amplification of cDNA Ends (RACE) Rapid amplification of cDNA ends (RACE) is a variation of RT-PCR that amplifies unknown cDNA sequences corresponding to the 3 - or 5 -end of the RNA (Hubank and Schatz, 1994). Numerous variations of the original protocols have been published (Troutt et al., 1992; Edwards et al., 1991; Edwards, 1993; Liu and Gorovsky, 1993; Fromont-Racine et al., 1993; reviewed in Schaefer, 1995) but will not be discussed in detail here. Two general RACE strategies exist: one amplifies 5 cDNA ends (5 RACE) and the other captures 3 cDNA end sequences (3 RACE). In either strategy, the first step in the RACE reaction involves the conversion of RNA into single-stranded cDNA using a reverse transcriptase. For the subsequent amplification reaction, two PCR primers are designed to flank the unknown sequence. One PCR primer is complementary to known sequences within the gene, and a second primer is complementary to an “anchor” site (anchor primer). The anchor site may be present naturally, such as the poly(A) tail of most mRNAs, or can be added in vitro after completion of the reverse transcription step. The anchor primer can also carry adaptor sequences, such as restriction enzyme recognition sites, to facilitate subsequent cloning of the amplified product. Amplification using these two PCR primers results in a product that spans the unknown 5 or 3 cDNA sequence, and sequencing this product will reveal the unknown sequence. The information obtained from partial cDNA sequences can then be used to assemble the sequence of the full-length cDNA (Frohman et al., 1988; Loh et al., 1989; Ohara et al., 1989).

9.4.11 AFLP Amplified Fragment Length Polymorphism (AFLP) is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci using a minimal number of PCR reactions (Vos et al., 1995). AFLP is composed of 3 steps:

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1. Cellular DNA is digested with one or more restriction enzymes. Typically this involves a combination of two restriction enzymes: a 4 base cutter (MseI) and a 6 base cutter (EcoRI). Ligation of linkers (restriction half-site specific adaptors) to all restriction fragments. 2. Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences. 3. Electrophoretic separation and amplicons on a gel matrix, followed by visualisation of the band pattern. The aim of this tool is to perform a theoretical AFLP-PCR experiment by using the same principles, and to suggest the adaptors and primers needed in the experiment.

9.4.12 In Situ PCR In situ PCR, first described in 1990, combines the sensitivity of PCR or RT-PCR amplification with the cellular or histological localization associated with in situ hybridization techniques (Haase et al., 1990). These features make in situ PCR a powerful tool for detecting proviral DNA, oncogenesis and localization of rare messages. The technique is amenable to analysis of fixed cells or tissue cross-sections. Detection of amplified products can be accomplished indirectly by subsequent hybridization using either radiolabeled, fluorescently labeled or biotin-labeled nucleic acid probes. PCR products can also be detected directly by the incorporation of a labeled nucleotide, although this method is subject to higher background levels. The use of in situ PCR requires altering some of the reaction parameters typical of basic PCR (Nuovo et al., 1993; Thaker, 1999). For example, increased Mg2+ concentrations (approximately 4.5 mM versus the normal 1.5–2.5 mM) are used for in situ PCR. An increased amount of DNA polymerase is also required unless BSA is added to the reaction, presumably because the polymerase binds to the glass plate and coverslip.

9.5 Cloning PCR Products 9.5.1 T-A Cloning Strategy Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated addition of a single nucleotide to the 3 -ends of PCR products. In the presence of all 4 dNTPs, dA is preferentially added; however, use of a single dNTP in a reaction mix results in (relatively inefficient) addition of that nucleotide. This complicates cloning, as the supposedly blunt-ended PCR product often is not, and blunt-ended cloning protocols often do not work or are very inefficient. This can be remedied by incubation of PCR products with T4 DNA pol or Klenow pol, which “polishes” the ends due to a 3 →5 exonuclease activity. However, this terminal transferase activity is also the basis of a clever cloning strategy: this uses Taq

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pol to add a single dT to the 3 -ends of a blunt-cut cloning vector such as pUC or pBluescriptTM, and simple ligation of the PCR product into the now “sticky-ended” plasmid.

9.5.2 Incorporation of Restriction Sites in Primers Although this may be rendered simple by incorporating the same or different restriction sites at the 5 -ends of PCR primers which allows generation of sticky ends and straightforward cloning into appropriate vectors – these should have at least two additional bases 5 to the recognition sequence to ensure that the enzymes will in fact recognise the sequence – and it is often found that even when this is done, the efficiency of cutting of fresh product is next to zero. This can sometimes be remedied by incubating fresh product with Proteinase K (to digest off tightly-attached Taq pol), but often is not. A solution to the problem is to use the “Klenow-Kinase-Ligase” (KKL) method: this involves “polishing” products with Klenow, kinasing them to get 5 -phosphorylation, ligating the fragments together to get concatemers, then restricting these with the appropriate restriction enzymes to generate the sticky-ended fragments suitable for cloning.

9.6 PCR as a Diagnostic Tool The Human Genome Project is a worldwide endeavor to map the DNA base sequence of every gene in the human genome. As of February 2001, the total number of functional genes is considerably less than expected, about 30,000 genes per cell compared with previous estimates of 100,000 genes. It has been estimated that a human somatic cell contains about 5 billion base pairs. If the average gene contains 1,500 bases, then 30,000 functional genes is only about 1% of the total DNA per cell. Although there is an estimated six feet of DNA per human cell, only a small fraction of this amount consititutes the actual protein-coding genes. PCR can be used in the analysis of disease genes by being able to amplify detectable amounts of specific fragments of DNA. The amplified fragments from disease genes may be larger, due to insertions, or smaller, due to deletions. The dramatic amplification of DNA by PCR allows the analysis of disease genes in extremely small samples of DNA. For example, only a small number of fetal cells need be extracted from amniotic fluid in order to analyze for the presence of specific disease genes. Additionally, single point mutations can be detected by modified PCR techniques such as the ligase chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP) analysis. The PCR technique also can be used to identify the level of expression of genes in extremely small samples of material, e.g. tissues or cells from the body. PCR is an extremely valuable technique in forensic criminology involving rape, murder and disputed parentage. DNA can be identified from small samples of blood,

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saliva, skin, hair follicles and semen. When amplifying genes using PCR, it is imperative that the sample not be contaminated with any foreign DNA, otherwise the foreign genes may be inadvertently amplified. PCR is also used for genetic testing to determine whether patients can carry a genetic mutation that could be passed on to their children (e.g. the mutation that causes cystic fibrosis) or to determine disease risk in patients themselves (e.g. a mutation in the gene BRCA1 predisposes a women to breast or ovarian cancer). Much of the DNA of humans is referred to as “variable number tandem repeats” (VNTRs) rather than specific protein-coding genes. The greatest variation in the DNA of two individuals is not in the protein-coding genes, but in the nonproteincoding sections of their DNA. Natural selection has resulted in some time-tested DNA sequences called genes which are identical in normal individuals. The exact number and order of amino acids in protein molecules are determined by the DNA base sequences of genes, and genetic mutations are essentially “misspelled” genes. Genetic mutations, including variations in the base sequences of vital genes, may be fatal if they fail to code for a vital enzyme. For example, the dominant gene for hemoglobin is a time-tested sequence of DNA bases that is essential for the production of this life-giving pigment. Hemoglobin is a quaternary protein composed of four polypeptides and 484 amino acids. The substitution of valine for glutamic acid (glutamate) in the beta polypeptide changes the oxygen-carrying potential of this vital blood cell pigment, and is the biochemical explanation for the genetic disease called sickle-cell anemia. PCR has a major place in human diagnostics, and is gradually emerging as a tool in plant diagnostics, and is now a routine tool in epidemiological and ecological studies, and for genetic mapping – RAPD and AFLP. The molecular diagnostic laboratory is responsible for the development and performance of molecular diagnostic tests for nucleic acid targets found in a variety of settings in medicine. PCR can become an important tool for medical diagnosis (Table 9.1). PCR can detect and identify bacteria and viruses that cause infections such a tuberculosis, chlamydia, viral meningitis, viral hepatitis, HIV, cytomegalovirus and many others. Using various types of PCR, the three broad areas of testing are genetics, hematopathology, and infectious disease. PCR is a powerful and reliable technique for rapid diagnosis of M. tuberculosis (Baek et al., 2000). The usefulness of PCR in the diagnosis of TB by using a variety of unselected clinical specimens is not clear as studies have differed in techniques including lysing method and target nucleic acid to detect products as well as the number and type of samples used, making the reported sensitivities and specificities difficult to compare (Pahwa and Hedau, 2005). A recent study comparing four conventional techniques, FNA cytology, ZN staining, culture and lymph node biopsies, and TB PCR indicated 94.8% diagnosis but PCR was found to be highly sensitive (94.4%) though less specific (38.2%) (Goel et al., 2001). Two types of JC virus (JCV) are found in infected brain and kidney tissues. A highly reliable allele-specific PCR is used to detect point mutations in cellular genes. (Ault et al., 1994).

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Table 9.1 Molecular diagnostic tests using PCR currently performed in a standard laboratory Genetics Factor V Leiden (R506Q) mutation MTHFR A233V mutations Prothrombin G20210A mutation Human platelet antigen (HPA0) (P1A1 and P1A2 ) Hemochromatosis (HFE) C282Y and H63D mutations Cystic fibrosis (DF508) mutation

Allele-specific PCR PCR-RFLP PCR-RFLP Allele-specific PCR PCR-RFLP

APC resistance Hyperhomocysteinemia Hypercoagulability Coronary artery thrombosis Iron overload

Allele-specific PCR

Newborn screening

PCR PCR

B cell clonality B cell clonality

Hematopathology IgH gene rearrangement T-cell receptor gene rearrangement, gamma/beta chains t(9;22) (BCR/ABL) Infectious disease Epstein-Barr virus Epstein-Barr virus Bacterial identification

RT-PCR

Diagnosis of CML

PCR In situ hybridization PCR/DNA sequencing

Quantitative Presense in tissue

Virology Adenovirus

PCR (TaqMan)

CMV DNA

PCR (TaqMan)

EBV DNA Enterovirus RNA HBV DNA HIV-1 RNA HIV-1 DNA HIV-1 resistance genotyping

HSV DNA Human metapneumovirus Influenza A and B Influenza A H5N1 Norovirus genogroups I and II Parvovirus B19 Polyomavirus BK

PCR (TaqMan) RT-PCR (TaqMan) PCR; Roche RT-PCR; Roche PCR; Roche TruGene sequencing; Bayer RT-PCR; Roche Invader assay; Third Wave PCR (TaqMan) RT-PCR (TaqMan) RT-PCR (TaqMan) RT-PCR (TaqMan) RT-PCR (TaqMan) PCR (TaqMan) PCR (TaqMan)

Qualitative and quantitative Qualitative and quantitative Qualitative Qualitative Quantitative Quantitative Qualitative Detects drug resistance mutations Quantitative Qualitative

Polyomavirus JC virus VZV DNA

PCR (TaqMan) PCR (TaqMan)

HCV RNA HCV genotype

Qualitative Qualitative Qualitative Qualitative Qualitative Qualitative Qualitative and Quantitative Qualitative Qualitative

Diagnosis of bacterial disease has improved in recent years, due to the routine use of PCR in the clinical laboratory (Clarke, 2002). The multiplex PCR is a sensitive assay with the ability to detect a mean of one to two genome copy units per 100 μl clinical sample and allows non-culture-based diagnosis of the diseasecausing organism within 3 h. A fluorescence-based multiplex PCR was automated

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for the simultaneous detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in clinical samples from patients with suspected meningitis (Smith et al., 2004). Specific identification of Bacillus anthracis and differentiation from closely related Bacillus cereus and Bacillus thuringiensis strains is still a major diagnostic problem. Commercially available diagnostic kits targeting plasmid-markers cannot differentiate between B. anthracis, non-anthracis Bacillus species harbouring anthrax-specific virulence plasmids, and plasmidless B. anthracis strains. A specific TaqMan PCR assay was successfully designed targeting sequences of gene locus BA 5345 of the B. anthracis strain based on a chromosomal marker. In another recent study by Lee and his collegues (2009) the multiplex real-time PCR assay was developed that could sensitively detect Salmonella spp. and specifically differentiate S. typhimurium from S. enteritidis in meats. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method is used for the detection and identification of Campylobacter, Helicobacter and Arcobacter species (Epsilobacteria) in clinical samples and evaluates its efficacy on saliva samples from humans and domestic pets. The PCR-DGGE method allows determination of the true prevalence and diversity of Epsilobacteria in clinical and other samples. Contact with the oral cavity of domestic pets may represent a route of transmission for epsilobacterial enteric diseases. Using “hot start” polymerase chain reaction (PCR) the distribution pattern of human papillomavirus (HPV) in vulvar lesions was analysed. After amplification, in situ analysis showed that many of the cells that lacked halos contain HPV DNA and that the hybridization signal often localized to areas where there was a thickened granular layer. HPV DNA was not noted in the basal cells. The one copy of HPV 16 was detectable after PCR with a single primer pair by in situ analysis only if the hot start modification was employed (Nuovo et al., 1992). Gong et al. (2002) employed nested PCR for diagnosis of tuberculous lymphadenitis and PCR-SSCP for identification of rifampicin resistance in fine-needle aspirates. Real-Time PCR can be applied to traditional PCR applications as well as new applications that would have been less effective with traditional PCR. A fluorogenicprobe hydrolysis (TaqMan)-reverse transcriptase (RT) PCR has been used for evaluation of diagnostic sensitivity of classical swine fever virus using clinical samples (Risatti et al., 2005). With the ability to collect data in the exponential growth phase, the power of PCR has been expanded into applications such as viral quantitation, pathogen detection including CMV detection, rapid diagnosis of meningococcal infection, penicillin susceptibility of Streptococcus pneumoniae, Mycobacterium tuberculosis and its resistant strains, waterborne microbial pathogens in the environment, genotyping (allelic discrimination) by melting-curve analysis or specific probes/beacons, Trisomies, single-gene copy numbers, microdeletion genotypes, haplotyping, quantitative microsatellite analysis, prenatal diagnosis/sex determination using single cell isolated from maternal blood or fetal DNA in maternal circulation and prenatal diagnosis of haemoglobinopathies intraoperative cancer diagnostics (Wittwer, 2001). Linear-after-the-exponential (LATE)-PCR is a new

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method for real-time quantitative analysis of target numbers in small samples. It is adaptable to high throughput applications in clinical diagnostics, biodefense, forensics, and DNA sequencing. Real-time PCR technology may also detect reciprocal duplications of common micro deletions. Duplications might be under-ascertained either due to incapacity of conventional PCR to detect duplications (as opposed to deletions), or because they could be associated with a mild, or absent, phenotype. The AZFa and AZFc duplications have already been identified as reciprocal deletion/duplication syndrome, and have been shown to be compatible with male fertility. For the rapid, simple and reproducible detection of antibiotic resistance genes in strains of methicillinresistant Staphylococcus aureus (MRSA) and Enterococcus, colony PCR using intact cells as the template DNA source in colony PCR was found efficient (Tsuchizaki, 2000). Pulsed-field gel electrophoresis and colony PCR were applied for the first time by Plourde-Owobi et al. (2005) for the molecular characterization of Clostridium tetani. Among five strains tested, one turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene. Genetic analysis of organisms (animals, plants and bacteria) at the molecular level is an important and widely practiced area of genetic science. A number of techniques developed over more than a decade offers the opportunity to identify each individual or type of individual in a species uniquely and unambiguously. PCR has impacted this area of analysis because of its ease of use and simplicity over traditional VNTR- and RFLP-based methods (Jeffreys et al., 1985; Sambrook and Russell, 2001). One important PCR-based genetic analysis is random amplified polymorphic DNA analysis (RAPD; reviewed in McClelland and Welsh, 1994; Power, 1996; Black, 1993). RAPD uses small, nonspecific primers for the amplification of regions of genomic DNA. Successful primer pairs produce different banding profiles of PCR products between individuals, strains, cultivars or species when analyzed by gel electrophoresis. Slight modifications to the basic PCR method are made for RAPD. First, the primers are approximately 10 bases in length compared to 17–23 base primer length of normal PCR. Because the primers are shorter, the temperature of the annealing reaction is reduced to less than 40◦ C. As with most PCR techniques, RAPD requires very little material for analysis and is relatively insensitive to the integrity of the material. No blotting techniques are required, thus eliminating the use of 32 P, bypassing probe generation and decreasing the amount of time required to obtain results.

9.7 Conclusion The use of PCR in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become a essential tool in research laboratories. Although conventional

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diagnostic techniques remain the method of choice in regions with low resource, PCR may be employed in cases with strong clinical suspicion and equivocal results, especially at an early stage of the disease for better diagnosis, management and treatment.

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Chapter 10

In Situ Hybridization

Abstract Nucleic acid hybridization techniques have contributed significantly to the understanding of gene organization, regulation and expression. In situ hybridization is a method for detecting specific nucleotide sequences by using a labeled complementary nucleic acid probe. The power of in situ hybridization can be greatly extended by the simultaneous use of multiple fluorescent colors. Multicolor fluorescence in situ hybridization (FISH), in its simplest form, can be used to identify as many labeled features as there are different fluorophores used in the hybridization. Key methodological advances have allowed facile preparation of low-noise hybridization probes, and technological breakthroughs now permit multi-target visualization and quantitative analysis – both factors that have made FISH accessible to all and applicable to any investigation of nucleic acids. In the future, this technique is likely to have significant further impact on live-cell imaging and on medical diagnostics. Keywords In situ hybridization · Clinical diagnostics · Chromosome · Fluorescence · FISH · Nucleic acid probe · Nucleic acid hybridization · Chromosome painting · Cytogenetics · End labeling · Probe

10.1 Prologue Nearly a quarter-century has passed since the first research articles introducing in situ hybridization as a method of detecting and studying DNA sequences in chromosomes and cells appeared in the literature (Gall and Pardue, 1969). In situ hybridization presents a unique set of problems as the sequence to be detected will be at a lower concentration, be masked because of associated protein, or protected within a cell or cellular structure. Therefore, in order to probe the tissue or cells of interest one has to increase the permeability of the cell and the visibility of the nucleotide sequence to the probe without destroying the structural integrity of the cell or tissue. In situ hybridization (ISH) with radiolabelled probes is a long established method in cytogenetics. This is distinct from immunohistochemistry, which localizes proteins in tissue sections. The technique of in situ hybridization is vital M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_10,

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to molecular biologists and their understanding of the pathophysiology of cellular functions.

10.2 The Concept of In Situ Hybridization “In situ” means “in the original place” in Latin, so ISH involves a labeled nucleic acid probe hybridizing with a DNA or RNA sequence in situ (in the cells). Normal hybridization requires the isolation of DNA or RNA, separating it on a gel, blotting it onto nitrocellulose and probing it with a complementary sequence. The basic principles for in situ hybridization are the same, except one is utilizing the probe to detect specific nucleotide sequences within cells and tissues (Hougaard et al., 1997). The sensitivity of the technique is such that threshold levels of detection are in the region of 10–20 copies of DNA/mRNA per cell.

10.2.1 The Process There are almost as many methods for carrying out in situ hybridization as there are tissues that have been probed. So more important than having a method is to have an understanding of the different stages in the process and their purpose (Litchter et al., 1988). 10.2.1.1 Preparation of Material The most common tissue sections used with in situ hybridization are frozen sections, paraffin embedded sections and cells in suspension. Cells can also be cytospun onto glass slides and fixed with methanol. 10.2.1.2 Choice of Probe Probes are complimentary sequences of nucleotide bases to the specific mRNA sequence of interest. These probes can be as small as 20–40 base pairs or be up to 1,000 bp. The strength of the bonds between the probe and the target plays an important role. The strength decreases in the order RNA-RNA to DNA-RNA. This stability is in turn influenced by the various hybridization conditions such as concentration of formamide, salt concentration, hybridization temperature, and pH. 10.2.1.3 Probe Types There are essentially four types of probes that can be used for performing in situ hybridization. Correctly designed and purified oligonucleotide probes will represent the easiest, fastest, least labor intensive and most inexpensive method. Oligonucleotide probes are produced synthetically by an automated chemical

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synthesis. The method utilizes readily available deoxynucleotides which are economical, but of course requires that you know the specific nucleotide sequence you wish to prepare (Hougaard et al., 1997). These probes have the advantages of being resistant to RNases and are small, generally around 40–50 base-pairs. This is ideal for in situ hybridization because of its stability, availability, faster and less expensive to use, easier to work with, more specific, better tissue penetration, better reproducibility and a wide range of labeling methods that do not interfere with target detection. The other probes are single stranded DNA probes, double stranded DNA probes and RNA probes (cRNA probes or riboprobes). They require time to prepare, expensive reagents are used during their preparation and a good repertoire of molecular skills is required for their use. 10.2.1.4 Labeling the Oligonucleotide The probe is hybridized (bound) within the tissue section or within cells and thus determines where the target gene is being expressed (Fig. 10.1). This can be detected by attaching to the probe an easily detectible substance or “labeling” before hybridization. Classically oligonucleotide probes have either 5 or 3 end-labeled or 3 tailed with modified nucleotides that have a “radiolabel” attached that can be

Fig. 10.1 The molecular mechanism of hybridization and detection by in situ hybridization technique

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detected after the probe has hybridized to its target. With end-labeling a single modified ddNTP (that incorporates the label) is added to either the 5 or the 3 end of the molecule enzymatically or during probe synthesis. 3 tailing involves addition of a tail (on average 5–50 nucleotides long of modified dNTPs depending on the method used) using the enzyme terminal transferase (TdT). The degree of specificity to which the probe hybridizes to the target sequence can be controlled by the design of the probe and the conditions of the buffer solution, including temperature, pH, and salt concentration. Hybridization mixtures usually have a small volume (about 10–20 μl total) with 50% formamide and hybridization typically occurs between 37–60◦ C (Palotie et al., 1996). 10.2.1.5 Detection As mentioned, radiolabeled probes are detectable using either photographic film or photographic emulsion using the technique of autoradiography. While in situ hybridization is undoubtedly a very powerful technique, for the average laboratory it is expensive to undertake, is time consuming, requires detailed molecular biological knowledge of sub cloning, in vitro transcription and bacterial expression (Hopman et al., 1986).

10.3 Disadavantages of Radioactive Probes In the early versions of in situ hybridization the probe was radioactively labeled probe but this procedure was unsatisfactory. With radioactive label it is difficult to achieve sensitivity and resolution, the two critical requirements for successful hybridization. Sensitivity requires that the radioactive probe has high emission energy, but if the radiolabel has high emission energy then it scatters its signal and gives poor resolution. High resolution is possible if a radiolabel with low emission energy, such as 3H, is used, but these have such low sensitivity that lengthy exposures are needed leading to a high background and difficulties in discerning the genuine signal (Landegent et al., 1987).

10.4 Solving the Problem: Advent of FISH Technique Over the past 15 years, however, a revolution in light microscopy has occurred through the development of fluorescence techniques that allow unprecedented ease, precision, and accuracy in locating, identifying, and recording data on the genetic makeup of biomedical samples. The problem with in situ hybridization with the use of radioactive probes was solved in the late 1980s by the development of nonradioactive, fluorescent DNA labels. These labels combine high sensitivity with high resolution and are ideal for in situ hybridization. Fluorescence in situ hybridization, the assay of choice for localization of specific nucleic acids sequences in native context, is a 20-year-old technology that has developed continuously. Over its

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maturation, various methodologies and modifications have been introduced to optimize the detection of DNA and RNA. The power of in situ hybridization can be greatly extended by the simultaneous use of multiple fluorescent colors. Multicolor fluorescence in situ hybridization (FISH), in its simplest form, can be used to identify as many labeled features as there are different fluorophores used in the hybridization enabling the relative positions of the probe sequences to be mapped. By using not only single colors, but also combinations of colors, many more labeled features can be simultaneously detected in individual cells using digital imaging microscopy. The pervasiveness of this technique is largely because of its wide variety of applications and the relative ease of implementation and performance of in situ studies. Although the basic principles of FISH have remained unchanged, high-sensitivity detection, simultaneous assay of multiple species, and automated data collection and analysis have advanced the field significantly. The introduction of FISH surpassed previously available technology to become a foremost biological assay.

10.4.1 Fluorescence In Situ Hybridization Fluorescence in situ hybridization is a molecular cytogenetic technique in which fluorescently labeled DNA probes are hybridized to metaphase spreads or interphase nuclei to detect or confirm gene or chromosome abnormalities that are generally beyond the resolution of routine cytogenetics. FISH have much higher rates of sensitivity and specificity. FISH also provides results more quickly because no cell culture is required. The first application of fluorescence in situ detection came in 1980, when RNA that was directly labeled on the 3 end with fluorophore was used as a probe for specific DNA sequences (Bauman et al., 1980). Enzymatic incorporation of fluorophore-modified bases throughout the length of the probe has been widely used for the preparation of fluorescent probes; one color is synthesized at a time (Wiegant et al., 1991). The use of amino-allyl modified bases, which could later be conjugated to any sort of hapten or fluorophore, was critical for the development of in situ technologies because it allowed production of an array of low-noise probes by simple chemistry (Wiegant et al., 1991). Methods of indirect detection allowed signal output to be increased artificially by the use of secondary reporters that bind to the hybridization probes. In the early 1980s, assays featuring nick-translated, biotinylated probes, and secondary detection by fluorescent streptavidin conjugates were used for detection of DNA (Manuelidis et al., 1982) and mRNA (Singer and Ward, 1982) targets. Approximately a decade later, improved labeling of synthetic, single-stranded DNA probes allowed the chemical preparation of hybridization probes carrying enough fluorescent molecules to allow direct detection. Many variations on these themes of indirect and direct labeling have since been introduced, giving a wide spectrum of detection schemes from which to choose (Pachmann, 1987; Rudkin and Stollar, 1977); the specifications, sensitivity and resolution of these techniques are well described elsewhere (Raap, 1998).

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Fig. 10.2 The schematic representation of fluorescence in situ hybridization technique

The technique of in situ hybridization is used to locate the chromosomal location of a specific DNA (or RNA) probe (Femino et al., 1998). The theory is the same as for Southern hybridization, except that the DNA to which the probe will hybridize is the actual chromosome. The probe is labeled with a flourescent probe. The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured a process that separates the complimentary strands within the DNA double helix structure (Fig. 10.2). The fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal. If the species has been characterized cytogenetically, the marker can be assigned to the appropriate chromosome. Because this technique

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uses a fluorescent probe it is called fluorescence in situ hybridization or FISH. It is also called chromosome painting.

10.4.2 FISH in Action Chromosome analyses by FISH have led to marked progress in cytogenetics research (Pinkel et al., 1986). A prime example of the power of hybridization approaches in genome investigation is comparative genomic hybridization, during which deletions and duplications of chromosomal regions are detected by differential fluorescence signals (Forozan et al., 1997). However, because the assay does not benefit from preservation of tissue structure or cellular architecture, its future applications are more likely to be in silico than in situ (Lichter et al., 1988). Initially, RNA assays could reliably detect only rather abundant messages, using clone-derived probes (Lawrence et al. 1989). Enhancements in detection and computer processing algorithms have subsequently allowed detection of smaller and smaller targets (Tanke et al., 1995; Piper et al., 1990; 1994). Advances in microscope and detector hardware have allowed the low light level produced by FISH to be recorded and analyzed with increasing sensitivity (Tanke et al., 1999). Mathematical image-processing algorithms have built on this process to yield super-resolution technology to probe at submicroscopic resolution, using digital image stacks (Carrington et al., 1995). New targets led to new applications of the FISH procedure. The new avenues of research opened by these applications required that more and more species be simultaneously detected (Fauth and Speicher, 2001). A major milestone in the detection of chromosome targets was the discrimination of all human chromosomes simultaneously, using computed interpretation of a 5-color scheme (Galbraith et al., 1991) Speicher et al., 1996). Although mRNAs can also be visualized in a multiplex fashion (Levsky et al., 2003), FISH analysis of the entire transcriptome in situ is a daunting task. One can only speculate that future technologies will feature increasingly higher-order multiplexing, until the number of interesting nucleic acid targets is reached. The technical means for color coding such a large number of entities is already in place (Nederlof et al., 1989, 1990) although reduction to practice will be difficult and a means of deciphering spatially overlapping signals will need to be developed. 10.4.2.1 FISH with Metaphase Chromosome FISH was originally used with metaphase chromosomes. These chromosomes, prepared from nuclei that are undergoing division are highly condensed with each chromosome in a set taking up a recognizable appearance characterized by the position of centromere and the banding pattern that emerges after the chromosome preparation is stained. With metaphase chromosomes, a fluorescent signal obtained by FISH is mapped by measuring its position relative to the end of the short arm of the chromosome.

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A disadvantage is that the highly condensed nature of the metaphase chromosomes means that only low resolution mapping is possible, two markers needing to be at least 1 Mb apart to be resolved as separate hybridization signals. This degree of resolution is insufficient for the construction of useful chromosome maps, and the main application of metaphase FISH has been in determining which chromosome a new marker is located on, and providing a rough idea of its map position, as a preliminary to a finer scale mapping by other methods. For several years these “other methods” did not involve any form of FISH, but since 1995 a range of higher resolution FISH is not restricted to intact chromosomes. It also works well on interphase nuclei, where it is used to visualize the order of closely spaced (from 100 to 500 kb apart) probes on a chromosome and to measure their distances apart. 10.4.2.2 FISH with Extended Chromosomes and DNA Fibers The resolution of FISH is determined not by the hybridization technique itself but by the nature of the chromosomal preparation being studied. If metaphase chromosomes are too condensed for fine scale mapping then chromosomes that are more extended be used. Mechanically stretched chromosomes can be obtained by modifying the preparative method used to isolate chromosomes from metaphase nuclei. Centrifugations generate shear forces which can result in the chromosomes becoming stretched upto 20 times their normal length. Individual chromosomes are still recognizable and FISH signals can be mapped in the same way as with normal metaphase chromosomes. The resolution is significantly improved and markers that are 200–300 Kb apart can be distinguished. Nonmetaphase chromosomes can be used (Lawrence et al., 1989; Siebert et al., 1998) because it is only during metaphase that chromosomes are highly condensed, at other stages of the cell cycle the chromosomes are naturally unpacked. Attempts have been made to use prophase nuclei as in these the chromosomes are still sufficiently condensed for individual ones to be identified. In practice these preparations provide no advantage over mechanically stretched chromosomes. Interphase chromosomes are more useful, as this stage of cell cycle (between nuclear divisions) is when the chromosomes are most unpacked. Resolution down to 25 Kb is possible, but chromosome morphology is lost so there are no external reference points against which to map the position of the probe. This technique is therefore used after preliminary map information has been obtained usually as a means of determining the order of a series of markers in a small region of a chromosome. Most excitingly, FISH can be used on stretched extended DNA fibers where it has a resolution of less than 5 kb.

10.5 Applications of FISH as a Diagnostic Tool for Research The development of in situ technologies has provided us with a wealth of information regarding the locations and expression patterns of genes in single cells. In situ hybridization (ISH) is an invaluable tool for research and diagnostics, dramatically

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advancing the study of cell- and tissue-specific expression of many genes. Some of ISH applications include determination of chromosome structure, function, and evolution, chromosomal gene mapping, expression of genes, localization of viral DNA sequences, diagnosis of viral diseases (Ahtiluoto et al., 2000), and localization of oncogenes (Suciu et al., 2008) sex determination and determination of chromosomal abnormalities (Siffroi et al., 2000; Brown et al., 2000). The uses and different approaches for ISH continue to increase, thus impacting many different research fields. New labeling techniques for probes, new detection systems, and advanced computer software increase the availability and efficiency of ISH (Leitch et al., 1994). More complete gene expression profiles of single cells will provide a new level of insight into the correlation of gene expression patterns with particular cellular phenotypes. This will be particularly important in studies of development and disease progression, where complicated; finely demarcated gene expression programs are in play.

10.5.1 To Analyze the Onset of Specific Gene Expression This technique has been utilized by neuroscientists to analyze the onset of specific gene expression during nervous system morphogenesis and to assess the cellular distribution of mRNAs encoding many important neuronal proteins. In the past decade, a number of genes related to nervous system induction, specification, and regionalization have been identified. In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs (∼20–24 nucleotides in length) presents difficulties for conventional in situ hybridization methods (Thompson et al., 2007).

10.5.2 Analysis of the Chromosome Structure The FISH technique is widely used in analysis of the chromosome structure, but is also gaining ground in areas as the experimental and routine karyotype analysis (Macville et al., 1997) of tumors. The method can be fine tuned to screen single tumor cells for genetic aberrations, as well as identify candidate tumor suppressor genes. Fluorescence in situ hybridization was used to map leukemia-related deletions of the long arm of chromosome 6 (6q) (Sinclair et al., 2004). Fluorescence in situ hybridization of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH (Hultdin et al., 1998) using a fluoresceinlabeled peptide nucleic acid (PNA) (CCCTAA) probe and DNA staining with propidium iodide.

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10.5.3 Localisation of RNA Transcripts Another application of in situ hybridisation is the localization of RNA transcripts in tissue, in analogy to the histochemical analysis of proteins. Probes that is labeled either radioactively or optically are hybridized with the RNA of a histological tissue section or preparation.

10.5.4 FISH as a Molecular Cytogenetic Technique to Understand Diseases Cytogenetics analysis is at present the basic element of the diagnostic process of genetic disorders which are caused by chromosomal abnormalities. Since the chromosome banding technique has been introduced in the 1970s, it has been available as a diagnostic tool of a number of clinical syndromes. However, since banding resolution is not always sufficient for the identification of chromosomal abnormalities, additional techniques for solving diagnostic dilemma of classical cytogenetics are needed. The basic method of molecular cytogenetics is fluorescence in situ hybridization. It enables a specific detection of unique sequences, chromosomal regions or entire chromosomes in metaphase, interphase cells or in tissue sections. Molecular cytogenetic approaches based on fluorescence in situ hybridization with chromosome-specific probes have been increased in recent years. They become a powerful tool for chromosomal diagnosis in prenatal, constitutional and cancers genetic disorders. In fact, various procedures are now available for rapid identification of numerical and structural chromosome aberrations that escape to conventional chromosome banding analysis. Fluorescence in situ hybridization is a molecular cytogenetic technique in which fluorescently labeled DNA probes are hybridized to metaphase spreads or interphase nuclei. Applications include identification of structurally abnormal chromosomes, including several of the cancer translocations, such as BCR/ABL and TEL/AML1 translocations; identification of marker chromosomes; detection of very small deletions (microdeletions); and rapid detection of Down syndrome and other numerical chromosome abnormalities; and the rapid detection of sex chromosomes and the SRY gene. FISH should be used in conjunction with G-banded chromosome analysis. FISH is also utilized to confirm microdeletions identified during high resolution chromosome analysis and to aid in identification of abnormal chromosomes. Interphase FISH is especially useful in bone marrow/cancer analyses when there is poor or no growth of the specimen (Tubbs et al., 2007). Chromosomal abnormalities are responsible for a considerable number of birth defects, and more than 50% of spontaneous abortions. These numbers have been significantly higher since the advent of FISH technology that allows the detection of submicroscopic chromosome alterations. 10.5.4.1 Microdeletion Syndromes A number of genetic syndromes are caused by the deletion of a small region of a particular chromosome. Often these deletions are too small to be picked up by standard

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or high resolution chromosome analysis, in which case, microdeletion syndrome probes must be used to elucidate the chromosome abnormality. These probes are pieces of DNA specific for the region deleted in the specified syndrome, and usually include a control probe which identifies the chromosome of interest. Wolf-Hirschhorn (4p-); Cri-du-chat (5p-);Williams syndrome (7q11.23); PraderWilli syndrome (15q11.2-q13); Angelman syndrome (15q11.2-q13); Miller-Dieker syndrome (17p13.3); Smith-Magenis syndrome (17p11.2); DiGeorge and Velocardio-facial syndromes (22q11.2); Kallman syndrome (Xp22.3); Steroid Sulfatase Deficiency (STS) (Xp22.3); X-Linked Ichthiosis (Xp22.3); Retinoblastoma (13q14); Trisomy Detection and Sex Determination; Probes for chromosomes 13, 18, 21, X, Y and SRY. These probes are used to screen interphase (uncultured) cells for trisomy 13, trisomy 18, trisomy 21, chromosome number for sex chromosomes (X and Y) and for the presence of the male determining gene SRY. FISH can be performed as an initial screening test in certain high risk situations where trisomy is suspected, such as in amniocytes from a pregnancy with an abnormal ultrasound or uncultured lymphocytes from an infant with ambiguous genitalia. 10.5.4.2 FISH For Oncology Screening FISH analysis is available to rule out certain common oncology related translocations, deletions and amplifications. This analysis is particularly useful when a specific hematologic disease is highly suspected (i.e. Philadelphia chromosome in chronic myelogenous leukemia) and/or cells fail to grow in culture. FISH can be used to look for minimal residual disease in patients undergoing treatment or in patients thought to be going into or coming out of remission. It can be used to detect unusual variants of the Philadelphia chromosome translocation, and to follow bone marrow transplants in certain patients. Since most of these oncology probes are used on interphase cells, standard cytogenetics is still necessary to look for other chromosomal aberrations and to detect clonal evolution of disease, an important prognostic indicator. Single Gene Probes (deletion or amplification) P58 CLK-1 Locus (1p36) D7S486 (7q31) Retinoblastoma (13q14) p53 (17p13.1) Her-2/neu (17q11.2-q12) Enumeration probes for all chromosomes Dual Color Translocation Probes Bcr/abl translocation t (9; 22) (q34; q11.2) (both major and minor breakpoints) M-bcr/abl translocation t (9; 22) (q34; q11.2) (major breakpoint) IGH/CCND1 translocation t (11; 14) (q13; q32) PML/RARA translocation t (15; 17) (q22; q21.1) TEL/AML1 translocation t (12; 21) (p13; q22) Amniotic Fluid

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Aneuploid detection by FISH for chromosomes 13, 18, 21, X and Y may be indicated in situations where there is a risk of a numerical chromosome abnormality. This risk is usually based on abnormal ultrasound findings. FISH may also be indicated in cases of late gestational age when a rapid result is required. FISH should be followed by a complete karyotype analysis and no clinical action should be taken based solely upon FISH results. FISH is best performed prior to 22 weeks gestation. After 22 weeks, considerable cellular debris is present and may lead to inconclusive results. FISH assay also provides a feasible and sensitive tool for the routine detection of the translocation t (8; 14) in interphase cells which might also offer new insights into the biology of high-grade B-cell lymphomas (Siebert et al., 1998). This translocation t (8; 14) (q24; q32) is the characteristic chromosomal aberration of Burkitt’s-type lymphomas and leukemias (BLs). As to the diagnostic and clinical relevance of the classical Burkitt translocation, the diagnosis of t (8; 14) is an important aim in the management of patients with BL. 10.5.4.3 FISH Diagnosis of Prenatal, Post Natal and New Born Diseases The clinical application of FISH technology in postnatal, prenatal, and preimplantation diagnoses has been playing an important role in the diagnosis and prevention of birth defects. As new technologies evolve, more and more new FISH techniquessuch as subtelomeric FISH, multicolor FISH (M-FISH), comparative genomic hybridization (CGH), and microarray are used in clinical diagnoses, the role of FISH technology in both research and clinical aspects of birth defects will surely continue to expand. Rapid aneuploidy detection methods allow prenatal diagnosis results to be released within 48 h, but not on the same day as the invasive test. A rapid fluorescence in situ hybridization (FISH) method (FastFISH) that releases accurate results on the same day as amniocentesis was developed recently (Choolani et al., 2007). Fast FISH was optimized to be completed within 2 h of sample collection using CEP and LSI probes for chromosomes 13, 18, 21, X, Y and DiGeorge syndrome (DGS). 10.5.4.4 FISH in Diagnosing Neoplasia Fluorescence in situ hybridization has become an important tool for diagnosing neoplasia in children. With probes designed to identify specific chromosomes and chromosomal regions, FISH is commonly used to detect the specific chromosomal abnormalities associated with hematologic diseases and solid tumors. Variations of FISH currently being investigated, such as comparative genomic hybridization, multicolor FISH, and microchip arrays, will probably result in additional uses of FISH in both research and clinical cytogenetic laboratories. Although FISH has disadvantages when compared with conventional cytogenetics and molecular methods, FISH will continue to be important in analyzing chromosomal abnormalities of tumors in children. Molecular probes for FISH are used for diagnosis of pediatric neoplastic diseases.

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10.5.5 FISH for Detection of Pathogens Human herpes virus 6 (HHV-6) DNA was detected in pontine nuclei of the brain cell of a patient by in situ hybridization (Ahtiluoto et al., 2000). In another study, Forrest et al. (2006) reported identification of Candida albicans in blood isolates by peptide nucleic acid fluorescence in situ hybridization (PNA FISH). This technique was 100% sensitive and specific in the rapid identification of 31 out of 72 candidemias as C. albicans and resulted in a significant reduction of caspofungin usage, with an overall cost savings of $1,729 per patient. In food, detection and identification of Salmonella species with the use of fluorescent in situ hybridization with 23S rRNAtargeted oligonucleotide probes was also reported by Qiang et al. (2003) and Fang et al. (2003).

10.6 Recent Advances of In Situ Hybridisation Technology Lately, fluorescence in situ hybridization technology has introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). A clarified cerotype for K562 obtained by a combination of the following molecular cytogenesis techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH was reported recently. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1; 18). Multicolor FISH further confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6), der(17)t(9;17), der(21)t(1;21), der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers. Using fluorescent in situ hybridization, three human BAC clones), localized in the terminal region of human chromosome 17p (HSA17p13; 1.44–3.68 Mp), were mapped to chromosome 8p of American mink (MVI8p) (Zhdanova, 2007). Reciprocal chromosome painting and hybridizations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes. Recent advances in fluorescence in situ hybridisation using probes specific for entire chromosomes or chromosome segments can quickly and economically provide a cytogenesis map of homologies (Lawrence et al., 1988). These probes cover large segments on chromosomes of various species and confirm previous hypotheses of extensive linkage conservation in mammals. Cytopathological diagnosis of Merkel cell carcinoma using interphase fluorescent in situ hybridization analysis was performed for research purposes using

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centromeric probes of chromosomes 6 and 8 (Suciu et al., 2008). Trisomy of chromosome 6 was found in 85% of tumour cells in the first case of MCC and case 2 exhibited trisomy 8 in 77% of tumour cells. In the absence of specific molecular markers, detection of trisomy 6 and/or trisomy 8 could help in identifying MCC. FISH analysis is easily and quickly performed on interphase nuclei may be extended to the study of other relevant genetic abnormalities. Recently using FISH analysis gene copy number alterations of circulating tumor cells in metastasis prostrate cancer patients have been reported (Leversha et al., 2009). FISH assay may be a promising tool for diagnosis, surveillance and monitoring of carcinoma in the upper urinary tract (Luo et al., 2009).

10.7 Conclusion The recently developed tissue microarray technology is an ideal platform for the introduction of high-throughput molecular profiling of tumor specimens at the single cell level. To construct a tissue microarray, small core biopsies are taken from representative areas of paraffin-embedded tumor tissues and assembled in a single block. Microtome sections are taken from the tissue microarray and placed on glass slides for rapid and efficient molecular analyses. In addition to pathological specimens such as tumor tissues, microarrays generally contain corresponding normal tissue and internal controls. The entire group of samples is analyzed simultaneously in one experiment, providing enormous amounts of correlative information about specific biomarkers, in the context of rigorous procedural controls. The next challenge will be to apply multi-gene FISH technology to these samples to correlate putative genes of prognostic value with specific morphological features initially, and then extend studies to samples where the morphology is not sufficiently informative. Certain genes can then be associated with the pre-cancerous state, for instance. Through such developments, one can foresee how molecular pathology could eventually surpass the limitations of morphological pathology. FISH has already colored the way that we visualize and conceptualize genes, chromosomes, transcription and nucleic acid movements. What remains to be seen is how exhaustive molecular analysis of single cells and tissue samples will impact how we identify, diagnose and alter the course of genetic pathology. Over the long term, it is expected that databases correlating gene expression patterns on the single cell level will accumulate as investigators and industries employ the technology of FISH with their favored biomarkers. Ultimately, FISH will be the preferred approach to anticipate the complicated components of gene expression leading to disease. These features should ensure greater use of in situ hybridization techniques in future research studies (Smith, 1997). When the tools become available for us to visualize multiple gene expression patterns in living cells, we will finally be able to fulfill the promise of FISH technology by building and testing models of molecular transcriptional dynamics within the true native context of the cell.

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Singer, R.H., Ward, D.C., 1982, Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc Natl Acad Sci USA 79:7331–7335. Smith, M.D., Triantafillou, S., Parker, A., Coleman, M., 1997, A nonradioactive method of in situ hybridization that uses riboprobes and paraffin-embedded tissue and its combination with immunohistochemistry. Diagnostic Mol Pathol 6(1). Speicher, M.R., Gwyn Ballard, S., Ward, D.C., 1996, Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 12:368–375. Suciu, V., Botan, E., Valent, A., Chami, L., Spatz, A., Vielh, P., 2008, The potential contribution of fluorescent in situ hybridization analysis to the cytopathological diagnosis of Merkel cell carcinoma. Cytopathology 19:48–51. Tanke, H.J., Florijn, R.J., Wiegant, J., Raap, A.K., Vrolijk, J., 1995, CCD microscopy and image analysis of cells and chromosomes stained by fluorescence in situ hybridization. Histochem J 27:4–14. Tanke, H.J., Wiegant, J., van Gijlswijk, R.P., Bezrookove, V., Pattenier, H., Heetebrij, R.J., Talman, E.G., Raap, A.K., Vrolijk, J., 1999, New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary Ratio labelling. Eur J Hum Genet 7:2–11. Thompson, R.C., Deo, M., Turner, D.L., 2007, Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes. Methods 43:153–161. Tubbs, R.R., Hicks, D.G., Cook, J., Downs-Kelly, E., Pettay, J., Hartke, M.B., Hood, L., Neelon, R., Myles, J., Budd, G.T., Moore, H.C., Andresen, S., Crowe, J.P., 2007, Fluorescence in situ hybridization (FISH) as primary methodology for the assessment of HER2 status in adenocarcinoma of the breast: a single institution experience. Diagn Mol Pathol 16:207–210. Wiegant, J., Ried, T., Nederlof, P.M., van der Ploeg, M., Tanke, H.J., Raap, A.K., 1991, In situ hybridization with fluoresceinated DNA. Nucl Acids Res 19:3237–3241. Zhdanova, N.S., 2007, Comparative mapping of mink (Mustela vison) chromosome 8p: localization of three human BAC clones. Genetika 43:1074–1078.

Chapter 11

Immunoassay

Abstract Antibody-based detection systems for specific Ags are versatile and powerful tools for various molecular and cellular analyses, as well as clinical diagnostics. The power of such systems originates from the considerable specificity of Abs for particular antigenic epitopes. There are, however, numerous examples where important biological markers for cancer, infectious disease, or biochemical processes are present at too low a concentration in body fluids or tissues to be detected by using conventional immunoassays. Keywords Antigen · Antibody · Immunoassay · ELISA · Radioimmunoassay · Immunogenicity · Immunoassays · Antigen-antibody interactions · Enzyme-linked immunosorbant assay · ImmunoRCA · nanoDLSA · Antibody-based microarray techniques · Protein microchips

11.1 Prologue Immunogenicity has always been an important consideration in the evaluation of pharmaceutical protein biologics (Geng et al., 2005). Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates. Currently available guidance documents dealing with the validation of bioanalytical methods address immunoassays in only a limited way (Findlay et al., 2000).


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11.2 Concept All assay procedures which do not rely on the direct detection of a specific property of the unknown substance must depend on the reaction of an unknown with a reagent to give a product which can then be assayed. The product can be estimated directly or indirectly (for example, as a result of a further reaction). If the unknown is regenerated after the primary reaction it may then react with more reagent and a cycling assay is established. The advantage of such a system is that one molecule of the unknown gives rise to several molecules of product with a consequent increase in sensitivity. The reagent can be as diverse as a dye, neutron irradiation, an enzyme substrate or an antibody. Adequate assay systems must be shown to have suitable specificity, sensitivity, precision, range and convenience. In order to obtain maximum sensitivity and precision: (1) all the unknown should be reacted at least once and preferably several times; (2) the amount of product should be assayed by a procedure which gives a low background and shows changes in direct proportion to the change in product concentration; (3) the property measured should be capable of detection at very low concentrations of the product. The use of one or more cycling reactions is one well recognized way of achieving suitable amplification. An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody to its antigen. Monoclonal antibodies are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules (Sipponen et al., 1976; Uotila, 1981). The antibodies picked must have a high affinity for the antigen (if there is antigen available, a very high proportion of it must bind to the antibody). Both the presence of antigen or antibodies can be measured. For instance, when detecting infection the presence of antibody against the pathogen is measured. For measuring hormones such as insulin, the insulin acts as the antigen. For numerical results, the response of the fluid being measured must be compared to standards of a known concentration. This is usually done though the plotting of a standard curve on a graph, the position of the curve at response of the unknown is then examined, and so the quantity of the unknown found. Detecting the quantity of antibody or antigen can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody. The label may consist of an enzyme such as enzyme immunoassay (EIA), radioisotopes such as I-125 (Radioimmunoassay – RIA), magnetic labels (magnetic immunoassay – MIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot.

11.3 Types of Immunoassay Several methods for the detection of an antibody response to a protein are described along with their limitations (Van Cleave, 2003). Immunoassays can be divided into those that involve labelled reagents and those which involve non-labelled

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reagents. Those which involve labelled reagents are divided into homogenous and heterogeneous (which involved a separation step) immunoassays. Heterogeneous immunoassays can be competitive or non-competitive. In a competitive immunoassay, the antigen in the unknown sample competes with labeled antigen to bind with antibodies. Whereas in noncompetitive immunoassays, the “sandwich assay,” antigen in the unknown is bound to the antibody site, then labeled antibody is bound to the antigen. The amount of labeled antibody on the site is then measured. Unlike the competitive method, the results of the noncompetitive method (Howell et al., 1981) will be directly proportional to the concentration of the antigen. Antigen-antibody interactions involving the formation of tertiary antibodyantigen-antibody complex has also been reported (Rubina et al., 2005). Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection. Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays can be recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin (Arenkov et al., 2000). The ELISA (enzyme-linked immunosorbant assay) is a powerful immunological method for detecting specific proteins in complex protein mixtures (Engvall et al., 1971; Von der Waat, 1978; Wolters et al., 1976). The ELISA has become an important tool for the cell and molecular biologist. It is increasingly being applied in clinical medicine for detecting proteins associated with disease including antibodies produced in response to infection by the HIV virus (Engvall and Perlmann, 1971). The ELISA is a fundamental tool of clinical immunology (Bosch et al., 1975; Engvall and Wewer, 2003; Mida et al., 2003; Schuurs and Van Weemen, 1980 Van weemen and schuurs 1974), and is used as an initial screen for HIV detection. Based on the principle of antibody-antibody interaction, this test allows for easy visualization of results and can be completed without the additional concern of radioactive materials use. ELISA measurement tool has also been used in parasitology e.g., malaria and trichinosis, microbiology, and oncology (Seppala et al., 1978); biochemistry of tissues, e.g., fibronectin, laminin, integrins, and muscular dystrophies. Currently, investigation using ELISA has been performed on the differentiation factors for muscle regeneration and myogenic cells from nonmuscle tissues for muscle cell replacement. During the late 1960s and early 1970s, many RIA test systems were essentially “home-brew” methods developed by individual researchers who could not keep pace (particularly financially) with the possibilities and facilities of commercial manufacturers such as Boehringer-Mannheim (Germany), Abbott (United States), and Organon Teknika (The Netherlands), to name only a few. Commercialization of EIA/ELISA test kits had started. Solid-phase techniques were used in the development of microtiter plates (96 wells) in which either an antigen or an antibody is

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noncovalently bound to a solid-phase support (Catt and Tregear, 1967). Technical advances led to automated pipetting devices (Micromedics; Hamilton), multichannel pipettes (Lab Systems), and microtiter plate readers and washers and in the 1980s fully automated test instruments were manufactured by BoehringerMannheim and Abbott, among others. Such automated systems have come to stay in medical laboratories. In the early 1970s, blood-bank screening for virologic diseases such as hepatitis B antigen was done either by (semi)automated RIA or nonradioactive but rather cumbersome hemagglutination tests. In 1976, Organon Teknika developed and marketed a highly successful EIA system for the hepatitis B surface antigen (HbsAg), featuring a 96-well microtiter plate format. This test became the first commercially available EIA. Other microbiological and virologic diagnostic tests soon followed, e.g., for hepatitis B “e” (HBe) antigens, rubella antibodies, toxoplasma antibodies, and in the 1980s, an EIA system for detection of human immunodeficiency virus antibodies.

11.4 Recent Advances in the Field of Immunodiagnostics Recent advances in the field of low-level Ag detection include the development of stronger fluorochromes and chemiluminescent substrates for use in ELISAs, immunofluorescence-based staining and immunoblotting, and the application of signal amplification methods such as tyramide deposition. Although these techniques can be quite powerful, greater sensitivity and specificity are often required, particularly when working with limited amounts of sample material or when Ag density is extremely low. With these needs in mind, rolling circle amplification (RCA) reporter system for the detection of protein Ags have been developed. RCA driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. Using a single primer, RCA generates hundreds of tandemly linked copies of the circular template within a few minutes. In ImmunoRCA, the 5 end of this primer is attached to an Ab. In the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction results in a DNA molecule consisting of multiple copies of the circle DNA sequence that remains attached to the Ab. The amplified DNA can be detected in a variety of ways, including direct incorporation of hapten-labeled or fluorescently labeled nucleotides, or by hybridization of fluor-labeled or enzymatically labeled complementary oligonucleotide probes (Schweitzer et al., 2000). ImmunoRCA, therefore, represents a novel approach for signal amplification of Ab–Ag recognition events. ImmunoRCA-profiling based on the simultaneous quantitation of multiple Ags should expand the power of immunoassays by exploiting the increased information content of ratio-based expression analysis. In immuno-PCR, a unique DNA sequence tag is associated with a specific Ab using streptavidin-biotin interactions, alternative bridging moieties, or covalent linkage. Abs bound to Ag are then detected by PCR amplification of the associated

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DNA tag. Multiple Abs and multiple DNA tags have been used to analyze several Ags simultaneously. Although immuno-PCR was shown to be significantly more sensitive than ELISA, gel electrophoresis was required after DNA amplification in solution to separate and/or quantitate the different amplified DNA tags. The requirements for thermal cycling and product separation by gel electrophoresis have restricted the widespread adoption of immuno-PCR as an alternative to ELISA and have precluded its utility in immunohistochemical or array formats. A research group has developed a highly sensitive homogeneous one-step immunoassay, nanoDLSA, for cancer marker detection. To prepare the immunoassay, a pair of monocolonal antibodies that can specifically bind with a cancer marker antigen, are conjugated with two gold nanoparticle probes. To conduct the assay, one simply mixes the two nanoprobes with a sample solution. The antigen-antibody binding will introduce a nanoparticle aggregation. By measuring the degree of nanoparticle aggregation in the assay solution using dynamic light scattering (DLS), the concentration of cancer marker antigen in the sample can be quantitatively determined. In a recent study, the research group has analyzed the free-PSA level of a few human serum samples collected from prostate cancer patients using both nanoDLSA and ELISA. The preliminary results revealed an excellent correlation between nanoDLSA and ELISA (Liu et al., 2008). The nanoDLSA immunoassay is fast, highly sensitive, accurate, and extremely easy to conduct. It requires a much smaller amount (at least 100 times less) of blood samples and antibody probes to conduct the assay compared to ELISA. The cost reduction of nanoDLSA compared to other immunoassays is tremendous. Because of the minute amount of sample that is required by nanoDLSA, it is possible to conduct the detection and measurement of one or multiple cancer markers from a single drop of human blood sample using this new immunoassay technology.

11.5 Clinical Applications of Immunoassay Most biopharmaceutical therapeutics elicits some level of antibody response against the product. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess immunogenicity of these molecules, appropriate detection, quantitation and characterization of antibody responses are necessary. Inadequately designed antibody assays have led to the hampering of product development or, during licensure, post-marketing commitments (Mire-Sluis et al., 2004). Antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment have been developed (Rucker et al., 2005). Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters

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for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples. The motor neuron degenerative disease spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and is caused by mutations in the survival of motor neurons (SMN) gene that reduce the expression levels of the SMN protein. A major goal of current therapeutic approaches is to increase SMN levels in SMA patients. Kolb et al., 2006 developed a novel cell immunoassay to quantitatively measure SMN levels from peripheral blood mononuclear cells (PBMCs) using a single anti-SMN antibody. In another study by Hix et al. (2004) a quantitative enzyme immunoassay (EIA) for detection of RBP (retinol-binding protein) was developed. An immunoassay for preß1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201 is useful for clinical measurement of preß1-HDL (Takashi et al., 2003). A newly developed enzyme-linked immunosorbent assay (ELISA) that detects immunoglobulin G antibodies to the 27-kDa Cryptosporidium parvum sporozoite surface antigen was used to test many sera collected from pregnant women (Ong et al., 2005). Serological assays may provide more accurate information regarding community levels of Cryptosporidium infection. Recently a commercial enzyme immunoassay PlateliaTM Dengue NS1 AG (BioRad Laboratories) was used to monitor semiquantitatively dengue virus replication in cultured cells (Ludert et al., 2008). These results suggest that the PlateliaTM Dengue NS1 AG kit can be used as a fast and reliable surrogate method for the relative quantitation of dengue virus replication in cultured cells. A immunoassay method may be suitable for determining levels of busulfan in human plasma. It offers the advantages of using a smaller sample size, does not require sample preparation and is less labor intensive than other methods. The ability to make 240 determinations per hour enables effective routine monitoring of busulfan levels in clinical practice. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates (Findlay et al., 2000). The double antigen bridging immunoassay

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has been used extensively for detection of immunogenicity responses to therapeutic monoclonal antibodies. A two-step format requires very low coating concentrations and higher conjugate concentrations to achieve maximal sensitivity and suffers from significantly reduced sensitivity at higher coating concentrations. A one-step assay format can greatly reduce the effect of coating concentration variation on assay performance (Bourdage et al., 2005). Protein microchips are used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements. A novel point-of-care platform to quantify micro-organisms causing dental infections and/or inflammatory markers reflecting an oral disease status has been studied by some workers. This system is based on a sandwich immunoassay technology known as ABICAP (Antibody Immuno Column for Analytical Processes) using poly-horseradish peroxidase conjugates. This assay enabled to quantify 500 colonyforming units of Streptococcus sobrinus per milliliter of saliva. The platform allows rapid and convenient performance chair side of such tests by a dentist or dental hygienist within 20 min at the dental office (Munial et al., 2007). An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Trichomonas vaginalis (a common sexually transmitted disease) which is both rapid and sensitive (detection limit of approximately 100 trichomonads per ml) has been developed. This assay employs affinity-purified rabbit anti-T. vaginalis antibodies in a “sandwich” configuration. It is simple to perform and is neither interfered with nor appears to cross-react with other microorganisms which are common inhabitants of the urogenital tract. In addition to exhibiting a sensitivity of 77%, the specificity of the ELISA was 100%. These results demonstrate that the ELISA is a significant improvement over the wet mount method for the diagnosis of trichomoniasis (Watt et al., 1986). Chronic fatigue syndrome in patients can be detected by immunoassay with cytomegalovirus early antigens from gene products p52 and CM2 (UL44 and UL57) (Beqaj et al., 2008).

11.6 Utilization and Interpretation of Immunological Tests Investigation plays an important role in diagnosis of clinical conditions. However, indiscriminate use of tests due to lack of knowledge of false positive and false negative test results, predictive value of a test etc. can cause more problem than benefit. It is imperative for the clinician to know, when to order a test, how to interpret it and what can be the methodological problems with the test. Cystic hydatid disease (hydatidosis) is one of the most important zoonosis that is caused by the larval stage of Echinococcus granulosus. As its diagnosis by clinical symptoms alone is difficult and confusing, serologic diagnostic techniques are used to confirm the disease. These techniques can also be used for epidemiologic studies. The commercial human enzyme-linked immunosorbent assay (ELISA) kit for the diagnosis of

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hydatidosis in sera collected from sheep with hydatidosis concluded that it is possible to use human ELISA kit for the diagnosis of hydatidosis in sheep (Hashemitabar, 2008).

11.7 Conclusion In conclusion, the number of analytical and clinical investigations relying on these measurement procedures worldwide is exceedingly large. Thus, one can imagine that the numbers of measurements and determinations using immunoassay for routine patient care are astronomical. The impact of diagnostic immunoassays on patients, clinicians, and the healthcare system in general is virtually overwhelming. Given the impact that the immuno assays had on clinical diagnosis and healthcare in general, as well as on the development of a well-established in vitro diagnostic industry, the inventors deserve to be honored again.

References Arenkov, P., Kukhtin, A., Gemmelll, A., Voloshchuck, S., Chupeeva, V., Mirzabekov, A., 2000, Protein microchips: use for immunoassay and enzymatic reactions. Ana Biochem 278(2): 123–131 Beqai, S.H., Lerner, A.M., Fitzgerald, J.T., 2008, Immunoassay with cytomegalovirus early antigens from gene products p52 and CM2 (UL44 and UL57) detects active infection in patients with chronic fatigue syndrome. J Clin Pathol 61(5):623–626. Bosch, A.M.G., Van Hell, H., Brands, J.A.M., Dijkhuizen, D.M., Schuurs, A.H.W.M., 1975, Methods for the determination of total estrogens (TE) and human placental lactogen (HPL) in plasma of pregnant women by enzyme-immunoassay. Clin Chem 21:1009. Bourdage, J.S., Lee, T.N., Taylor, J.M., Willey, M.B., Brandt, J.T., Konrad, R.J., 2005, Effect of double antigen bridging immunoassay format on antigen coating concentration dependence and implications for designing immunogenicity assays for monoclonal antibodies. J Pharm Biomed Anal 39(3–4):685–690. Catt, K., Tregear, G.W., 1967, Solid-phase radioimmunoassay in antibody-coated tubes. Science 158:1570–1572. Engvall, E., Jonsson, K., Perlmann, P., 1971, Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochim Biophys Acta 251(3):427–434. Engvall, E., Perlmann, P., 1971, Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874. Engvall, E., Wewer, U.M., 2003 The new frontier in muscular dystrophy research: booster genes. FASEB J;17:1579–1584. Findlay, J.W., Smith, W.C., Lee, J.W., Nordblom, G.D., Das, I., DeSilva, B.S., Khan, M.N., Bowsher, R.R., 2000, Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective J Pharm Biomed Anal 21(6):1249–1273. Geng, D., Shankar, G., Schantz, A., Rajadhyaksha, M., Davis, H., Wagner, C., 2005, Validation of immunoassays used to assess immunogenicity to therapeutic monoclonal antibodies. J Pharm Biomed Anal 39(3–4):364–375. Hashemitabar, G.R., Razmi, G.R., Shahroozian, A., 2008, Application of a modified human enzyme-linked immunosorbent assay kit for diagnosis of hydatidosis in sheep Iranian J Vet Res 9(1) serial no. 22:31–35.

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Hix, J., Martinez, C., Buchanan, I., Morgan, J., Tam, M., Shankar, A., 2004, Development of a rapid enzyme immunoassay for the detection of retinol-binding protein. Am J Clin Nutrition 79(1):93–98. Howell, E.E., Nasser, J., Schray, K.J., 1981, Coated tube enzyme immunoassay: factors affecting sensitivity and effects of reversible protein binding to polystyrene. J Immunoassay 2(3–4): 205–225. Kolb, S.J., Gubitz, A.K., Olszewski, R.F., Jr., Ottinger, E., Sumner, C.J., Fischbeck, K.H., Dreyfuss, G., 2006, A novel cell immunoassay to measure survival of motor neurons protein in blood cells. BMC Neurol 6:6. Liu, X., Dai, Q., Austin, L., Coutts, J., Knowles, G., Zou, J., Chen, H., Huo, Q., 2008, A onestep homogeneous immunoassay for cancer biomarker detection using glod nanoparticle probes coupled with dynamic light scattering. J Am Chem Soc 130(9):2780–2782. Ludert, J.E., Mosso, C., Ceballos-Olvera, I., del Angel, R.M., 2008, Use of a commercial enzyme immunoassay to monitor dengue virus replication in cultured cells. Virol J 5:51. Mida, T., Miyazaki, O., Nakamura, Y., Hirayama, S., Hanyu, O., Fukamachi, I., Okada, M., 2003, Analytical performance of a sandwich enzyme immunoassay for preβ-HDL in stabilized plasma. J Lipid Res 44(3):645–650. Mire-Sluis, A.R., Barrett, Y.C., Devanarayan, V., Koren, E., Liu, H., Maia, M., Parish, T., Scott, G., Shankar, G., Shores, E., Swanson, S.J., Taniguchi, G., Wierda, D., Zuckerman, L.A., 2004, Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J Immunol Methods 289(1–2):1–16. Munial, S., Miethe, P., Netuschil, L., Struck, F., Maier, K., Bauermeister, C., 2007, Immunoassaybased diagnostic point-of-care technology for oral specimen. Ann N Y Acad Sci 1098:486–489. Ong, C.S., Li, A.S., Priest, J.W., Copes, R., Khan, M., Fyfe, M.W., Marion, S.A., Roberts, J.M., Lammie, P.J., Isaac-Renton, J.L., 2005, Enzyme immunoassay of cryptosporidium-specific immunoglobulin G antibodies to assess longitudinal infection trends in six communities in British Columbia, Canada. Am J Trop Med Hyg 73(2):288–295. Rubina, A.Y., Dyukova, V.I., Dementieva, E.I., Stomakhin, A.A., Nesmeyanov, V.A., Grishin, E.V., Zasedateley, A.S., 2005, Quantitative immunoassay of biotoxins on hydrogel-based protein m microchips. Anal Biochem 340(2):317–329. Rucker, V.C., Havenstrite, K.L., Herr, A.E., 2005, Antibody microarrays for native toxin detection. Anal Biochem 339(2):262–270. Schuurs, A.H.W.M., Van Weemen, B.K., 1980, Enzyme-immunoassay: a powerful analytical tool [Review]. J Immunoassay 1:229–249. Schweitzer, B., Wiltshire, S., Lamber, J., O’Malley, S., Kukanskis, K., Zhu, Z., Kingsmore, S.F., Lizardi, P.M., Ward, D.C., 2000, Immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci USA 97(18):10113–10119. Seppala, M., Rutanen, E.M., Heikinheimo, M., Jalanko, H., Engvall, E., 1978 Detection of trophoblastic tumour activity by pregnancy-specific ß1 glycoprotein. Int J Cancer 21:265–267. Sipponen, P., Ruoslahti, E., Vuento, M., Engvall, E., Stenman, U., Ihamakit, T., Suirala, M., 1976, CEA and CEA-like activity in gastric cancer. Acta Hepatogastroenterol (Stuttg) 13:276–279. Takashi, M., Miyazaki, O., Nakamura, Y., et al., 2003, Analytical performance of a sandwich enzyme immunoassay for preß1-HDL in stabilized plasma, J Lipid Res 44:645–650. Uotila, M., Ruoslathi, E., Engvall, E., 1981, Two-site sandwich enzyme immunoassay with monoclonal antibodies to human alphafetoprotein. J Immunol Methods 42(1):11–15. Van Cleave, V.H., 2003, Vailidation of immunoassays for anti-drug antibodies. Dev Biol (Basel) 112:107–112. Van Weemen, B.K., Schuurs, A.H.W.M., 1974, Immunoassay using antibody-enzyme conjugates. FEBS Lett 43:215–218. Von der Waat, M., Snelting, A., Cichy, J., Wolters, G., Schuurs, A., Schuurs, A.H.W.M., 1978, Enzyme immunoassay in diagnosis of hepatitis with emphasis on the detection of “e” antigen (HbeAg). J Med Virol 3(1):43–49.

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Watt, R.M., Philip, A., Wos, S.M., Sam, G.J., 1986, Rapid assay for immunological detection of Trichomonas vaginalis. J Clin Microbiol 24(4):551–555. Wolters, G., Kuijpers, L.P.C., Kacaki, J., Schuurs, A.H.W.M., 1976, Enzyme-immunoassay for HbsAg. Lancet II:690.

Chapter 12

Phage Display

Abstract Phage display is a method of generating antibody reagents, and phageantibody libraries to provide a rich source of antibody diversity, potentially providing hundreds of unique antibodies per target. The antibody gene, once isolated, can be conveniently shuttled into a variety of expression formats, providing a renewable resource of antibody protein. Keywords Antibodies · Combinatorial libraries · Protein–protein interaction · Virions · Phage display · Epitope mapping · Cloning allergens · Drug discovery

12.1 Prologue The selection of antibodies from combinatorial libraries displayed on the surface of filamentous phage has become an important methodology for the generation of reagent, diagnostic, and therapeutic molecules and for the study of natural immune responses. Using this technique, antibody genes have been cloned from multiple species or expressed directly from large man-made repertoires of antibody-encoding genes.

12.2 Concept Phage display is the development of phage-displayed peptides and antibody fragments into viable diagnostic reagents (Jayanna et al., 2009). Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for almost any target (Fig. 12.1). A crucial advantage of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype, which allows the evolution of the selected binders into optimized molecules. Phage display facilitates engineering of antibodies with regard to their size, valency, affinity, and effector functions. The selection of antibodies and peptides from libraries displayed on the surface of filamentous phage has M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_12,

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Fig. 12.1 General concept of phage display

proven significant for routine isolation of peptides and antibodies for diagnostic and therapeutic applications (Azzazy and Highsmith, 2000). Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. Phage display is a practical realization of the artificial chemical evolution. Using standard recombinant DNA technology, peptides are associated with replicating viral DNAs that include the peptides’ coding sequences. The peptide populations so created are managed by simple microbiological methods. Phage display is an exponentially growing research area, and numerous reviews covering different aspects have been published in recent years.

12.3 Phage-Display Libraries as Populations of Replicable, Mutable Chemicals Phages are viruses that infect bacterial cells, and many of the vectors used in recombinant DNA research are phages that infect the standard recombinant DNA host: the bacterium Escherichia coli. The key feature of recombinant DNA vectors, including phages, is that they accommodate segments of “foreign” DNA-pieces of human DNA, for instance, or even stretches of chemically synthesized DNA. As vector DNA replicates in its E. coli host, then, the foreign “insert” replicates along with it as a sort of passenger. An “expression vector,” including a phage-display vector, has an additional feature compared to vectors in general: the foreign DNA is “expressed” as a protein. That is, it programs machinery of the E. coli host cell to synthesize a foreign peptide whose amino acid sequence is determined (via the genetic code) by the nucleotide sequence of the insert. Phage display differs from conventional expression systems,

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however, in that the foreign gene sequence is spliced into the gene for one of the phage coat proteins, so that the foreign amino acid sequence is genetically fused to the endogenous amino acids of the coat protein to make a hybrid “fusion” protein. The hybrid coat protein is incorporated into phage particles (“virions”) as they are released from the cell, so that the foreign peptide or protein domain is displayed on the outer surface. A phage-display “library” is a heterogeneous mixture of such phage clones, each carrying a different foreign DNA insert and therefore displaying a different peptide on its surface. Different types of libraries will be formed. Each peptide in the library can replicate. The phage to which the peptide is attached infects a fresh bacterial host cell and it multiplies to produce a huge crop of identical progeny phages displaying the same peptide. And if the phage’s DNA suffers a mutation in the peptide coding sequence that mutation is passed on to the phage’s progeny and can affect the structure of the peptide. In short, the peptides in a phage-display library have the two key characteristics required for chemical evolution: replicability and mutability. Because of its accessibility to solvent, a displayed peptide often behaves essentially as it would if it were not attached to the virion surface. This means that many techniques that chemists or biochemists apply to compounds free in solution can be applied more or less unaltered to peptides tethered to a phage. The captured phages are “amplified” by infecting them en masse into fresh cells and culturing the cells to yield a large crop of progeny phages, which can serve as the input for another round of affinity purification. Moreover, by periodically introducing mutations into the phage population, the experimenter widens the search for effective ligands by exploring peptide sequences that are not present in the initial phage-display library. Eventually, captured phages are cloned so that the displayed peptides responsible for binding can be studied individually. The amino acid sequence of the peptide is easily obtained by determining the corresponding coding sequence in the viral DNA. This so-called “affinity selection” is the premier example of artificial selection imposed on populations of phage-displayed peptides. Since there is no need to process clones one by one until the final stage, enormous libraries displaying billions of different structures can be easily surveyed for exceedingly rare binding clones. Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology’s success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. The utility of phage display as a powerful tool in modern biotechnology is well known (Sindhu, 2001).

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The selection of peptides and proteins from libraries expressed on the surface of filamentous phage is becoming an important tool in biotechnology. Recent developments have shown that peptides can be selected to bind receptors and antibodies, while semi synthetic antibodies can be selected to bind almost any target. Phage display has allowed the routine isolation of therapeutically interesting human antibodies. Phages are also being utilized to examine the specificities of natural enzymes as well as to evolve novel enzymes de novo (Barbas, 1993). Phage display technology presents a rapid means by which proteins and peptides that bind specifically to predefined molecular targets can be isolated from extremely complex combinatorial libraries. There are several important ways by which phage display can provide impetus to receptor-based research. Firstly, phage display can be applied, alongside transcriptome and proteome expression profiling techniques, to the identification and characterisation of receptors whose expression is specific to a cell lineage, a tissue or a disease state. Secondly, specific monoclonal antibodies that enable researchers to identify, localize and quantify receptors can be produced very rapidly (weeks). Thirdly, it should be possible to apply phage display to the matching of orphan ligands and receptors. Finally, phage display can be used to identify proteins and peptides that modulate receptor activity. As well as being useful in the study of receptor function, biologically active proteins and peptides could also be used therapeutically, or as leads for drug design. Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural legends using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires (Faix et al., 2004). Hence phage display is ready to play a central role in the study of receptors in the post-genome era (Hartley, 2002).

12.4 Practical Applications of Phage Display Phage display is a simple yet powerful technology that is used to rapidly characterize protein-protein interactions from amongst billions of candidates. Usual targets for isolation of phage displayed peptide ligands include antibodies, various receptors, other full size proteins or larger fragments thereof. Smaller protein fragments such as synthetic peptides have not been reported as targets for screening of peptide display libraries (Bremnes et al., 1998). This widely practiced technique is used to map antibody epitopes, create vaccines and to engineer peptides, antibodies and other proteins as both diagnostic tools and as human therapeutics.

12.4.1 Target Receptors Used in Affinity Selection Over the past decade, powerful technologies devoted to survey large molecular libraries for the presence of specific clones using the discriminative power of affinity selection have been developed. Phage surface display technology is the most established of these methods and has revolutionised our ability to select agonistic

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and antagonistic peptides, antibodies with desired specificity and other drug targets. Thereby ligands are expressed as fusions to phage coat proteins and their respective genes are packaged within the phage. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Binding of the phage to the target molecule offers a selective system by which rare phage carrying the desired gene product can be selected from large phage populations carrying inappropriate sequences. Phage selected in this fashion can be used for subsequent rounds of selection because they are able to self-replicate in suitable host cells, yielding target-specific phage populations after several consecutive rounds of affinity selection (Rhyner et al., 2004).

12.4.2 Epitope Mapping and Mimicking An “epitope” is the small determinant on the surface of a ligand with which the receptor makes close, geometrically and chemically specific contact. If the ligand is a protein, the epitope is sometimes “continuous,” comprising a few adjacent critical amino acids in the primary sequence. For instance, antibodies specific for continuous epitopes on protein antigens typically contact three to four critical amino acids over a six-residue segment. More often, however, protein epitopes are more complex. Many are “discontinuous” because they comprise critical binding residues that are distant in the primary sequence but close in the folded native conformation. And many epitopes, including discontinuous ones, are “conformation-dependent” because they require the context of the overall protein structure to contrain them in a binding conformation. In many research contexts, it is highly desirable to “map” the epitope to a confined portion of the natural protein ligand. If the epitope is (or might be) continuous and not conformation dependent, random peptide libraries provide a cheap, easy approach to this goal. The receptor is used to affinity select random peptide ligands, and the sequence motif in the selected peptides is compared to the amino acid sequence of the natural ligand. Often, in these cases, the motif clearly matches critical binding amino acids in the natural protein ligand, thereby mapping the epitope to a very narrow part of the overall natural ligand structure. Since this approach uses replicable, widely available, all-purpose random peptide libraries and simple microbiological procedures, it is generally much cheaper and easier than alternative epitope mapping methods that require chemical synthesis of short peptide segments of the ligand’s amino acid sequence. Only rarely will a random peptide library contain a binding motif extending to more than about six amino acids or adequately represent conformation-dependent or discontinuous epitopes. Although receptors recognizing such epitopes often select ligands from random peptide libraries, these artificial ligands seldom bear a recognizable similarity to any part of the natural protein ligand at the amino acid sequence level. An alternative approach in such circumstances is to construct a gene-specific

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library displaying 15–100 amino acid segments of the natural amino acid sequencelong enough to occasionally include small elements of secondary structure from the native protein. Such libraries sometimes contain good ligands for receptors that fail to select ligands from random peptide libraries. Because it requires construction of a specific library for each new ligand gene, however, this approach is much more arduous than use of all-purpose random peptide libraries. Affinity selection from random peptide libraries often reveals entirely unexpected ligands-ligands that do not match any linear epitope and that could not have been anticipated from even extensive knowledge of the receptor and/or its natural ligand. This is especially so when the receptor’s natural epitope is nonproteinaceous or is a discontinuous or conformation-dependent protein epitope.

12.4.3 Identifying New Receptors and Natural Ligands A ligand for a receptor can be used as a “probe” to identify new receptors that bind the same ligand (Faix et al., 2004). Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. These methods will be useful as functional genomics tools for identifying natural ligands from various source tissues. The selection of phage displayed cDNA repertoires on an immobilized target has been reported to be an efficient way to rapidly identify interacting partners (Hufton, 1999). To date, however, only a few successful applications have been reported. These vectors may be used for selection of phage displayed cDNA libraries with polyclonal sera from patients. This will allow the identifying antigenic cDNA products in such diseases as cancer, viral/bacterial infections or autoimmune disease. Furthermore, by selections with other specific biomolecules, this display system may aid the identification of interacting partners in functional genomics.

12.4.4 Drug Discovery Many of the receptors used in affinity selection are targets of drug discovery programs, and the peptide ligands selected by them are therefore potential leads to new drugs. Such peptides might act as receptor agonists or antagonists (for example, of enzymes or hormone receptors) or otherwise modulate the receptor’s biological effect. Affinity selection resembles in essence the traditional approach to drug discovery: screening libraries of synthetic compounds or natural products for substances that bind the target receptor and that might therefore be leads to new agonists, antagonists, or modulators. There are important differences, however. Affinity selection has the key advantage that the scale of the search is many orders of magnitude greater than is feasible when chemical libraries must be screened compound by compound- billions of peptides versus tens of thousands of chemicals. On the other hand, for most pharmaceutical applications, peptides have poor pharmacological

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properties, being generally orally unavailable and subject to rapid degradation in the body by naturally occurring enzymes. There is some precedent for synthesizing peptidomimetic compounds that mimic the essential pharmacological features of bioactive peptides on a nonpeptide scaffold (Section VI). But developing peptidomimetics is an arduous and chancy project in medicinal chemistry, and it seems likely that the most important contribution of phage display to drug discovery will be confined to applications where peptides themselves can serve as plausible therapeutics. For example, Wrighton and colleagues used phage display to identify a small peptide agonist of the receptor for eythropoietin, a protein hormone that is administered parenterally in some circumstances. The small peptide, which bears little resemblance to the natural hormone at the amino acid sequence level, might serve as a superior substitute for the much larger protein. Vaccines are another case in which peptides are eminently usable therapeutics. Peptides composed of D-amino acids are much less susceptible to degradation in the body than peptides composed of the natural L-amino acids. Schumacher and his colleagues have put forth a clever way of using phage display to identify D-amino acid peptide ligands for target receptors. They synthesized chemically the D form of an SH3 domain and used it to affinity select ligands from a random peptide library, whose amino acids are of course the natural L isomers. The D forms of these peptides are therefore ligands for the natural L form of the receptor-the form that would be the actual target of drug discovery.

12.4.5 Epitope Discovery – A New Route to Vaccines and Diagnostics In recent years, the use of display vectors and in vitro selection technologies has transformed the way in which we generate ligands, such as antibodies and peptides, for a given target. Using this technology, we are now able to design repertoires of ligands from scratch and use the power of phage selection to select those ligands having the desired (biological) properties. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. A combination of the design and generation of millions to billions of different ligands, together with phage display for the isolation of binding ligands and with functional assays for identifying (and possibly selecting) bio-active ligands, will open even more challenging applications of this inspiring technology, and provide a powerful tool for drug and target discovery well into the next decade (Hoogenboom et al., 1998). When the receptor used for affinity selection is an antibody, the peptides it selects from random peptide libraries are called “antigenic mimics” of the corresponding natural epitope-the antigenic determinant that elicited the selector antibody in the first place. When these peptides are used in turn to immunize naive animals, some are able to elicit new antibodies that cross-react with the natural epitope, even though the naive animals have never been directly exposed to it. Such peptides are “immunogenic mimics” as well as antigenic mimics. Antigenic and immunogenic

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mimicry are the basis of “epitope discovery”, a new approach to disease diagnosis and vaccine development. Most diseases-particularly infectious diseases-leave their imprint on the complex mixture of antibody specificities that comprises the total serum immunoglobulin population. Included in this population are disease-specific antibodies-some elicited directly by antigens on a pathogen, others possibly recognizing antigens that reflect the disease process more indirectly. When total serum antibody from a patient is used to affinity select clones from a random peptide library, therefore, some of the selected ligands will correspond to disease-specific antibodies. Of course the patient’s pool of antibodies will contain myriad non-disease-specific antibodies, too, so it may require extensive counterselection or screening with antibodies from control subjects (not suffering from the disease) to identify those peptides that correspond to authentic disease-related antibody specificities and that therefore can be considered diagnostic for the disease. This is an eminently “portable” program of discovery, using the same procedure and the same “all-purpose” random peptide libraries regardless of the particular disease. Even in the most difficult cases, it nets a rich diversity of diagnostic peptides with far less work than is required to identify antigenic peptides by direct study of a pathogen’s antigenic makeup.

12.4.6 Selection of DNA-Binding Proteins Phage display may help molecular biologists realize a long-standing goal: to design proteins that specifically bind a given target DNA sequence (O’Neil and Hoess, 1995). Rational design has poor prospects in this field, since there do not seem to be simple rules of complementarily-comparable to those governing base-pairing between complementary single-stranded nucleic acids-by which the sequence specificity of a DNA-binding protein can be predicted from the amino acids at critical positions in its structure. A much more promising approach is to construct a library of randomized variants of a parent DNA binding domain (e.g., one of the zinc-finger domains, a common DNA-binding motif in eukaryotic nuclei) displayed on a filamentous phage; randomization is concentrated on positions that are thought to make sequence-specific contacts with the target DNA in the parent domain. From this library, clones that bind a new target DNA sequence, different from that recognized by the parent domain, are then affinity-selected. In an experiment analogous to epitope mapping, phage display has been used to map the DNA binding site of SATB1, a nuclear matrix protein that specifically binds the minor groove of a DNA sequence motif called MAR. Using an MAR DNA sequence as the immobilized receptor, affinity-selected peptides from a random peptide library; the predominant peptide shared 50% sequence identity with a nine-residue segment of the SATB1 sequence-a segment that was subsequently shown on independent grounds to be critical for DNA recognition. Phage display has also been used to affinity-select a hexapeptide with some binding preference for the single-stranded heptadeoxycytidilate (dC)7 , although in this case no mapping purpose was in view.

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12.4.7 Landscape Libraries as a Source of New Materials The surface landscape of a filamentous virion is a cylindrical array of thousands of repeating subunits composed of the exposed parts of the major coat protein pVIII; this exposed shell accounts for about half the weight of the particle. When a random peptide is displayed on every copy of this protein, it subtends a major fraction (20% or more) of the repeating unit and thus of the entire particle surface. Unless the random peptide is loosely tethered to the bulk of the major coat protein, it is forced to interact with residues in its immediate neighbourhood, and may therefore be constrained in a definite three-dimensional conformation that differs markedly from the surface conformation of wild-type particles and of clones displaying other random peptides. A large population of such clones can therefore be regarded as a library of “organic landscapes”. The ensemble of a random peptide in a landscape library with its surrounding wild-type residues may have emergent properties that are lost when the peptide is excised from its context. Such peptides are analogous to the complementarilydetermining regions of antibodies-oligopeptide loops that in the context of the intact protein make most of the specific contacts with antigen but as free peptides seldom have appreciable antigen-binding propensities. Localizable emergent properties are present even when the foreign peptide is displayed on only an occasional pVIII molecule, as in type 88 and 8+8 systems. Nevertheless, the high-density display in landscape phage may greatly enhance overall effectiveness in some applications. For instance, if a single target receptor complex can bind two or more neighbouring peptides on the phage surface, the overall effective affinity may be enhanced many orders of magnitude compared to monovalent binding. Some emergent properties are not localizable to a single subunit but seem instead to be a global property of the entire surface landscape. Thus, for instance, phage clones that are highly resistant to chloroform were selected from a landscape library; their entire surface is composed of hybrid pVIII subunits displaying a peptide motif that confers resistance to the solvent. In contrast, mosaic phage coated with roughly equal numbers of such hybrid subunits and wild-type subunits showed almost no resistance, indicating that resistance is not an additive property to which each hybrid subunit contributes independently. Landscape phage might be looked on as a new kind of submicroscopic “fiber.” Each phage clone is a type of fiber with unique surface properties. These fibers are not synthesized one by one with some use in mind. Instead, billions of fibers are constructed, propagated all at once in a single vessel and portions of this enormous population are distributed to multiple end-users with many different goals. Each user must devise a method of selecting from this population those fibers that might be suitable for his or her particular application-by affinity selection or whatever other selection principle ingenuity can conjure up. Localizable or global emergent properties cannot be transferred from the virion surface to another medium; any application that depends on such properties must therefore use phages themselves as the new material. This undoubtedly precludes some applications of phage “fibers”: it is doubtful we will be wearing clothes made

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of them, for instance. Still, filamentous phages are essentially proteins manufactured by a fermentation process and as such are potentially usable in any of the myriad of applications that might be contemplated for such proteins.

12.4.8 Phage Display-Combinatorial Chemistry on the Cheap The selection of antibodies from combinatorial libraries displayed on the surface of filamentous phage has become an important methodology for the generation of reagent, diagnostic, and therapeutic molecules and for the study of natural immune responses. Using this technique, antibody genes have been cloned from multiple species or expressed directly from large man-made repertoires of antibody-encoding genes. Recent studies demonstrate that the technique allows for the in vitro evolution of antibodies to create molecules whose affinity for antigen exceeds that observed in nature (Rader and Barbas, 1997). For drug discovery and a handful of other high-profile applications with high commercial stakes, phage display is perhaps not an optimal technology. For the ordinary research user, however, it has the overwhelming advantage that it is cheap and easy. It uses standard microbiological techniques that are familiar to all molecular biologists, and its key resources-phage libraries and clones-are replicable and therefore nearly cost-free after their initial construction or selection. It is astonishing to contemplate that within a single 1.5-ml micro centrifuge tube we can fit a few hundred trillion phage particles displaying billions of different peptide structuresabundance and diversity from which hundreds of different users with altogether different purposes in mind can select clones of great value.

12.4.9 Cloning Allergens by Phage Display Allergic diseases represent an immune disorder associated with the production of immunoglobulin E (IgE) against normally innocuous antigens (allergens). Almost 20% of the population in industrialized countries suffers from type I allergic symptoms such as allergic rhinitis, conjunctivitis, urticaria or asthma. Although the mechanisms responsible for these allergic reactions are quite well understood, knowledge about the repertoire of molecules able to elicit type I symptoms is still limited. To clone and characterize entire allergen repertoires from complex allergenic sources in a fast and efficient way, new technologies are required (Rhyner et al., 2004). Selective enrichment of cDNA libraries displayed on phage surface with serum IgE from allergic individuals combined with robotic-based highthroughput screening technology has proved to be extremely successful for the rapid isolation of allergens. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones using the discriminative power of affinity purification. The selection of IgE-binding clones involves the enrichment of phage binding

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to serum IgE immobilised to a solid phase during consecutive rounds of affinity selection. As a consequence of the physical linkage between genotype and phenotype, sequencing of the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the surface-displayed allergen. In spite of some biological limitations imposed by E. coli as expression host, phage surface display technology has strongly contributed to the rapid isolation of a vast variety of IgE-binding structures (Appenzeller et al., 2001). The screening of cDNA libraries displayed on phage surfaces with immobilized serum IgE from allergic patients reduces the time required for the selection of candidate clones to a few weeks. Robot-assisted high-throughput screening of the enriched library provides a fast and cost-effective way to isolate complete allergen repertoires. The biotechnological production of recombinant allergens derived from these sequences bears a high potential for the improvement of the diagnosis of allergic diseases. It provides valuable data when applied to the investigation of IgE interaction with allergens. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions (Davies et al., 2000). Phage display technology presents a rapid means by which proteins and peptides that bind specifically to predefined molecular targets can be isolated from extremely complex combinatorial libraries. There are several important ways by which phage display can provide impetus to receptor-based research. Firstly, phage display can be applied, alongside transcriptome and proteome expression profiling techniques, to the identification and characterization of receptors whose expression is specific to a cell lineage, a tissue or a disease state. Secondly, specific monoclonal antibodies that enable researchers to identify, localize and quantify receptors can be produced very rapidly (weeks). Thirdly, it should be possible to apply phage display to the matching of orphan ligands and receptors. Finally, phage display can be used to identify proteins and peptides that modulate receptor activity. As well as being useful in the study of receptor function, biologically active proteins and peptides could also be used therapeutically, or as leads for drug design. Hence phage display is ready to play a central role in the study of receptors in the post-genome era.

12.5 Conclusion Screening of phage-displayed libraries of proteins and peptides has, for nearly a decade, proven to be a highly effective method for finding much needed “needles” in a vast molecular “haystack”. Phage display of proteins has become an important tool for protein engineering. This technique has been used to solve an increasing diversity of problems, including identification of binding motifs for much smaller targets and the use of novel screening (Rodi and Makowski, 1999). New phage derived bio recognition nanomaterials have emerged recently as a result of the in depth study of the genetics and structure of filamentous phage and evolution of phage display

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technology (Petrenko, 2008). Recently a novel approach of nanocarrier targeting through their fusion with target specific phage coat proteins was reported by Jayanna et al. (2009). Over the past year, the versatility of the technology has expanded to include the development of DNA-binding proteins with novel specificities, energetics of protein folding and directed evolution of antibodies. In addition, display of expressed cDNA libraries opens an exciting opportunity for studying protein-protein interactions (O’Neil and Hoess, 1995).

References Appenzeller, U., Blaser, K., Crameri, R., 2001, Phage display as a tool for rapid cloning of allergenic proteins. Archivum immunologiae et therapiae experimentalis 49(1):19–25. Azzazy, H.M., Highsmith, W.E., Jr., 2000, Phage display technology: clinical applications and recent innovations. Clin Biochem 35(6):425–445. Barbas, C.F., III, 1993, Recent advances in phage display. Curr Opin Biotechnol 4(5):526–530. Bremnes, T., Lauvark, V., Lindqvist, B., Blakke, O., 1998, Selection of phage displayed peptides from a random 10-mer library recognising a peptide target. Immunotechnology 4:21–28. Davies, J.M., O’hehir, R.E., Suphioglu, C., 2000, Use of phage display technology to investigate allergen-antibody interactions. J Allergy Clin Immunol 105(6 Pt 1):1085–1092. Faix, P.H., Burg, M.A., Gonzales, M., Ravey, E.P., Baird, A., Larocca, D., 2004, Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection. Biotechniques 36:1018–1029. Hartley, O., 2002, The use of phage display in the study of receptors and their ligands. J Recept Signal Transduct Res 22(1–4):373–392. Hoogenboom, H.R., de Bruine, A.P., Hufton, S.E., Hoet, R.M., Arends, J.W., Rooyers, R.C., 1998, Antibody phage display technology and its applications. Immunotechnology 4(1):1–20. Hufton, S.E., Moerkerk, P.T., Meulemans, E.V., de Bruïne, A., Arends, J.W., Hoogenboom, H.R., 1999, Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands. J Immunol Methods 231(1–2):39–51. Jayanna, P.L., Torchilin, V.P., Petrenko, V.A., 2009, Liposomes targeted by fusion phage proteins. Nanomedicine 5(1):83–89. O’Neil, K.T., Hoess, R.H., 1995, Phage display: protein engineering by directed evolution. Curr Opin Struct Biol 5(4):443–449. Petrenko, V., 2008, Evolution of phage display: from bioactive peptides to bioselective nanomaterials. Expert Opin Drug Deliv 5(8):825–836. Rader, C., Barbas, C.F., III, 1997, Phage display of combinatorial antibody libraries. Curr Opin Biotechnol 8(4):503–508. Rhyner, C., Weichel, M., Fluckiger, S., Hemmann, S., Kleber-Janke, T., Crameri, R., 2004, Cloning allergens via phage display. Methods 32(3):212–218. Rodi, D.J., Makowski, L., 1999, Phage-display technology – finding a needle in a vast molecular haystack. Curr Opin Biotechnol 10(1):87–93. Sidhu, S.S., 2001, Engineering M13 for phage display. Biomol Eng 18:57–63.

Chapter 14

DNA Biosensors

Abstract As we enter into the twenty-first century, advances in medical technology help in the discovery of more genetic diseases. DNA sensing has become increasingly important for rapid genetic screening and detection. DNA biosensors offer considerable promise for obtaining the sequence specific information in human, viral and bacterial nucleic acid in a faster, simpler and cheaper manner as compared to traditional hybridization assays. The development of a DNA hybridization biosensor helps in wide scale ability of pathogen detection and molecular diagnostics. Keywords Biosensor · Food diagnostics · Environment monitoring · DNA biosensor · Diagnostics · Nanosensor · Immunosensor · Immobilisation · Bioreceptors · Analytes · DNA diagnostics · Electrochemical biosensors · Nanoparticles · Genosensors · Peptide nucleic acid · PNA · Miniaturization · Nanofabrication

14.1 Prologue Few scientific areas have witnessed dramatic changes of the magnitude observed recently in DNA diagnostics. With the completion of the human genome project (HGP), we are at the just beginning of a revolution in genetic analysis. The information obtained from the HGP opens a door to tremendous analytical opportunities ranging from diagnostics tests for mutations to the assessment of medical treatment. To continue these advances to exploit these opportunities and to address the growing market needs in the twenty-first century, future devices must link high performances, with speed, simplicity and low cost (Wang, 2002a). With advancement of miniaturization technologies, commercialization of such instruments for molecular diagnostics are now available for genetic testing. Biosensors have several potential advantages over other methods of analysis (Alocilja and Radke, 2003), including sensitivity in the range of ng/mL for microbial toxins and 95% and is 75% sensitive and 100% specific for neonatal meningitis. The rapid diagnosis of HSV encephalitis can prevent a brain biopsy and rapidly determine the need for acyclovir therapy. Enterovirus is the most common cause of aseptic meningitis in the summer and fall months in temperate climates and accounts for 10–20% of encephalitis cases. Nucleic acid amplification has also been successfully applied to other etiologies of viral CNS disease, such as CMV and varicella-zoster virus (VZV), but these assays have not been implemented as broadly as those for HSV and EV, so are still transitioning to becoming the method of choice. It should be noted that not all encephalitis viruses are readily detected by NAA. For example, due to the short period of viremia in many carboviral infections (i.e., West Nile Virus), CSF NAA has low sensitivity, and the gold standard remains serology. CSF NAA false negative results can occur due to collection of CSF very early or very late in illness, rapid viral clearance in immunocompetent hosts, and NAA inhibitors. False positive CSF NAA results also occur primarily due to lack of data to suggest the detection of certain viral nucleic acids correlates with clinical CNS disease, but can also be caused by the presence of peripheral blood in the CSF. While CSF NAA is considered by many the diagnostic standard of care as discussed above, the lack of

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standardized FDA-approved assays has made implementation of CSF NAA difficult in nonacademic settings. While most laboratories offering CSF NAA use qualitative methods, data indicate a role for quantitative CSF NAA in differentiating nonspecific presence of virus and virus-associated disease, to aid in prognosis for improved patient management, and in monitoring antiviral therapy.

19.3.4 Bacterial Infections The quest for the search of rapid, cheap, easy to perform and highly sensitive and specific tests has resulted into the introduction and application of various new tests for the diagnosis of bacterial meningitis. Most of these tests are based on immunological principles, viz. counter-immunoelectrophoresis, latex agglutination test, co-agglutination, radio-immunoassay, haemagglutination inhibition as well as study of immune profile of cerebrospinal fluid. Apart from these certain non-immunological tests viz. Limulus amoebecyte lysate test, gas liquid chromatography, nitroblue tetrazolium dye reduction test have also been evaluated to make the laboratory diagnosis of this important clinical entity rapid and more reliable (Ichhpujani and Bhatia, 1984) The tuberculin skin test for immunologic diagnosis of Mycobacterium tuberculosis infection has many limitations, including being confounded by Bacillus Calmette-Guérin (BCG) vaccination or exposure to nontuberculous mycobacteria. M. tuberculosis-specific antigens that are absent from BCG and most nontuberculous mycobacteria have been identified (Tiwari et al., 2007). Mori et al., (2004) examined the use of two of these antigens, CFP-10 and ESAT-6, in a whole blood IFN-gamma assay as a diagnostic test for tuberculosis in BCG-vaccinated individuals. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the _gt11 vector. DNA from a virulent M. tuberculosis strain was sheared and expressed to generate shotgun polypeptides. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These recombinant proteins when used in ELISA differentiated BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62% (Bisen et al., 2003). A simple, rapid and cost-effective glycolipid antibody based liposome agglutination assay viz., TB Screen Test, for diagnosis of patients with pulmonary and extrapulmonary tuberculosis with 94% sensitivity and 98.3% specificity was developed (Tiwari et al., 2005). The assay clearly differentiated healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. A TB/M card test based on liposomal agglutination test was developed by Tiwari et al. (2007) for detection of glycolipid antigens of M. tuberculosis. The test was shown to be effective in detecting glycolipid antigens of M. tuberculosis in clinical samples from patients with active TB with as

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low as 1 ng/ml analytical sensitivity, 97.4% clinical sensitivity and 96.9% specificity. The test was proved to be highly economical, rapid, simple and instrument free. The test seems fairly useful for mass testing of a variety of biological specimens (cerebrospinal, pleural and synovial fluids, serum, tissue biopsy extract) from patients with tuberculous meningitis, pulmonary TB and other extra-pulmonary TB in endemic countries. The sensitivity of NAA detection of MTB in smear-positive respiratory specimens is 96.9%, and the specificity is 100%, whereas the sensitivity and specificity in smear-negative specimens is 72.0 and 99.3%, respectively. It should be noted that NAA of MTB does not replace the need for routine mycobacterial culture and susceptibility testing. In addition to the direct detection of MTB, techniques such as probe-based technology and sequence analysis can be applied to cultured isolates to decrease the time to identification over routine biochemical analysis. Rapid identification of MTB impacts not only patient care, but also infection control. Due to the increasing frequency of isolation of mycobacterial species associated with immunocompromised hosts and the increased incidence of multi-drug resistant MTB, it has become imperative to offer accurate yet rapid diagnostic tools for the detection and identification of mycobacteria (Tiwari et al., 2007). A debate exists regarding the gold standard for the laboratory diagnosis of Bordetella pertussis. Historically, culture plates collected at the patient’s bedside (i.e., cough plates) have been considered the reference method. Although culture is very specific, its sensitivity suffers partially due to the organism’s fastidious nature, but primarily because the highest sensitivity for culture occurs before patients are symptomatic. NAA remains positive for longer after therapy than culture, and NAA is also positive for a longer period after onset of symptoms. Therefore, NAA is useful for patients presenting later in their illness. NAA testing allows for same-day results and since erythromycin-resistant B. pertussis is still rare, a cultured isolate is rarely needed for antimicrobial susceptibility testing. Multiple studies have demonstrated significant increased detection of B. pertussis when comparing NAA to culture: reported PCR-positive, culture-negative samples range from 13 to 88%. However, due to potential false positive and false negative results with B. pertussis NAA procedures, it is strongly recommended that results be considered in the context of patient clinical presentation, and clinically inconsistent results should be confirmed by a second method. NAA is also being used in bacteriology to detect antimicrobial resistance. Since antimicrobial resistance can be multi-factorial, this practice is limited to organisms in which the results can be interpreted with confidence in regard to the genotypic relationship to clinical treatment and/or infection control precautions. Such examples are direct detection of vancomycin-resistant Enterococcus (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) from rectal and nares surveillance cultures, respectively. Screening patients for VRE and MRSA carriage is a key strategy for preventing the spread of these organisms in health care settings. NAA technology reportedly increases VRE detection by up to 120%. In addition, enterococci that confer low-level intrinsic resistance, and thus not considered “true” VRE, are accurately ruled out preventing unnecessary contact precautions

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and contributing to hospital savings. NAA detection of MRSA has been shown to be equal in sensitivity to culture-based methods, but has the advantage of offering a faster turnaround time, thus impacting hospital cost savings. However, it should be noted that direct specimen testing for MRSA comes with limitations, often including a lower positive predictive value than conventional methods. More recently, new strains of MRSA have appeared that are associated with skin and soft tissue infections in outpatients and are called community-associated MRSA (CA-MRSA). The increasing incidence of CA-MRSA is causing overall rates of MRSA to rise. Therefore, it has become even more important to quickly and accurately identify resistant isolates.

19.3.5 Fungal Infections The incidence of invasive fungal infections has increased considerably in recent years. Mycetomas are chronic, granulomatous, subcutaneous infections caused by either actinomycetes bacteria or eumycetes fungi. The disease is endemic in the tropics and is characterised by a slow progression with risks of bone and visceral involvement. There have been notable advances in improved molecular techniques for species identification. Carbapenems, oxazolidinones and triazoles have emerged as promising therapeutic options, but access to drug therapies in developing countries remains limited by the poor availability and high costs (Ameen and Arenas, 2008). The molecular assay using PCR-ELISA help in the diagnosis of invasive fungal infections at the early stage of infection, before clinical manifestations (Badiee et al., 2009).

19.3.6 Practical Applications of Molecular Methods in the Clinical Microbiology Laboratory Commercial kits for the molecular detection and identification of infectious pathogens have provided a degree of standardization and ease of use that has facilitated the introduction of molecular diagnostics into the clinical microbiology laboratory (Table 19.1). The use of nucleic acid probes for identifying cultured organisms and for direct detection of organisms in clinical material was the first exposure that most laboratories had to commercially available molecular tests. Although these probe tests are still widely used, amplification-based methods are increasingly employed for diagnosis, identification and quantitation of pathogens, and characterization of antimicrobial-drug resistance genes. Commercial amplification kits are available for some pathogens (Table 19.1), but some clinically important pathogens require investigator-designed or “home-brew” methods (Table 19.2). In addition, molecular strain typing, or genotyping, has proven useful in guiding therapeutic decisions for certain viral pathogens and for epidemiologic investigation and infection control.

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19.3.6.1 Molecular Epidemiology Laboratory characterization of microbial pathogens as biologically or genetically related is frequently useful in investigations. Several different epidemiologic typing methods have been applied in studies of microbial pathogens (Table 19.4). The phenotypic methods have occasionally been useful in describing the epidemiology of infectious diseases; however, they are too variable, slow, and labor-intensive to be of much use in most epidemiologic investigations. Newer DNA-based typing methods have eliminated most of these limitations and are now the preferred techniques for epidemiologic typing. The most widely used molecular typing methods include plasmid profiling, restriction endonuclease analysis of plasmid and genomic DNA, Southern hybridization analysis using specific DNA probes, and chromosomal DNA profiling using either pulsed-field gel electrophoresis (PFGE) or PCR-based methods. All these methods use electric fields to separate DNA Table 19.4 Genotypic methods for epidemiologic typing of microorganismsa Method

Examples

Comments

Plasmid analysis

Staphylococci enterobacteriaceae

Restriction endonuclease analysis of chromosomal DNA with electrophoresis

Enterococci; Staphylococcus aureus Clostridium difficile; Candida spp

PFGE

Enterobacteriaceae; Staphylococci Enterococci; Candida spp. Enterobacteriaceae; Staphylococci Pseudomonas aeruginosa Mycobacterium tuberculosis Candida spp. Enterobacteriaceae Acinetobacter spp. Staphylococci M. tuberculosis HCV

Plasmids may be digested with restriction endonuclease, only useful when organisms carry plasmids Large number of bands difficult to interpret not amenable to computer analysis Fewer bands; Amenable to computer analysis very broad application Fewer bands; computer analysis Sequence-based profiles Automated

Genome restriction fragment length polymorphism analysis; ribotyping, insertion sequence probe fingerprinting PCR-based methods: repetitive elements PCR spacer typing, selective amplification of genome restriction fragments, multilocus allelic sequence-based typing Library probe genotypic hybridization schemes: multilocus probe dot-blot patterns, high-density oligonucleotide patterns a The

Burkholderia cepacia S. aureus M. tuberculosis High-density

Crude extracts and small amounts of DNA may suffice

Unambiguous yes-no result Less discrimination than other methods Couple with DNA chip technology

table contains examples of available methods and applications and is not intended to be all-inclusive; Adapted from Pfaller.

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fragments, whole chromosomes, or plasmids into unique patterns or fingerprints that are visualized by staining with ethidium bromide or by nucleic acid probe hybridization. Molecular typing is performed to determine whether different isolates give the same or different results for one or more tests. Epidemiologically related isolates share the same DNA profile or fingerprint, whereas sporadic or epidemiologically unrelated isolates have distinctly different patterns. If isolates from different patients share the same fingerprint, they probably originated from the same clone and were transmitted from patient to patient by a common source or mechanism. Molecular typing methods have allowed investigators to study the relationship between colonizing and infecting isolates in individual patients, distinguish contaminating from infecting strains, document nosocomial transmission in hospitalized patients, evaluate reinfection versus relapse in patients being treated for an infection, and follow the spread of antimicrobial-drug resistant strains within and between hospitals over time. Most available DNA-based typing methods may be used in studying nosocomial infections when applied in the context of a careful epidemiologic investigation. In contrast, even the most powerful and sophisticated typing method, if used indiscriminately in the absence of sound epidemiologic data, may provide conflicting and confusing information.

19.3.6.2 Financial Considerations Molecular testing for infectious diseases includes testing for the host’s predisposition to disease, screening for infected or colonized persons, diagnosis of clinically important infections, and monitoring the course of infection or the spread of a specific pathogen in a given population. It is often assumed that in addition to improved patient care, major financial benefits may accrue from molecular testing because the tests reduce the use of less sensitive and specific tests, unnecessary diagnostic procedures and therapies, and nosocomial infections. However, the inherent costs of molecular testing methods, coupled with variable and inadequate reimbursement by third-party payers and managed-care organizations, have limited the introduction of these tests into the clinical diagnostic laboratory. Not all molecular diagnostic tests are extremely expensive. Direct costs vary widely, depending on the test’s complexity and sophistication. Inexpensive molecular tests are generally kit based and use methods that require little instrumentation or technologist experience. DNA probe methods that detect C. trachomatis or N. gonorrhoeae are examples of low-cost molecular tests. The more complex molecular tests, such as resistance genotyping, often have high labor costs because they require experienced, well-trained technologists. Although the more sophisticated tests may require expensive equipment (e.g., DNA sequencer) and reagents, advances in automation and the production of less-expensive reagents promise to decrease these costs as well as technician time. Major obstacles to establishing a molecular diagnostics laboratory that are often not considered until late in the process are required licenses, existing and pending patents, test selection, and billing and reimbursement.

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Molecular screening programs for infectious diseases are developed to detect symptomatic and asymptomatic disease in individuals and groups. Persons at high risk, such as immunocompromised patients or those attending family planning or obstetrical clinics, are screened for CMV and Chlamydia, respectively. Likewise, all blood donors are screened for bloodborne pathogens. The financial outcome of such testing is unknown. The cost must be balanced against the benefits of earlier diagnosis and treatment and societal issues such as disease epidemiology and population management. One of the most highly touted benefits of molecular testing for infectious diseases is the promise of earlier detection of certain pathogens. The rapid detection of M. tuberculosis directly in clinical specimens by PCR or other amplification-based methods is quite likely to be cost-effective in the management of tuberculosis. Other examples of infectious disease that are amenable to molecular diagnosis and for which management can be improved by this technology include HSV encephalitis, Helicobacter pylori infection, and neuroborreliosis caused by Borrelia burgdorferi. For HSV encephalitis, detection of HSV in cerebrospinal fluid (CSF) can direct specific therapy and eliminate other tests including brain biopsy. Likewise, detection of H. pylori in gastric fluid can direct therapy and obviate the need for endoscopy and biopsy. PCR detection of B. burgdorferi in CSF is helpful in differentiating neuroborreliosis from other chronic neurologic conditions and chronic fatigue syndrome. As discussed earlier, molecular tests may be used to predict disease response to specific antimicrobial therapy. Detection of specific resistance genes (mec A, van A) or point mutations resulting in resistance has proven efficacious in managing disease. Molecular-based viral load testing has become standard practice for patients with chronic hepatitis and AIDS. Viral load testing and genotyping of HCV are useful in determining the use of expensive therapy such as interferon and can be used to justify decisions on extent and duration of therapy. With AIDS, viral load determinations plus resistance genotyping have been used to select among the various protease inhibitor drugs available for treatment, improving patient response and decreasing incidence of opportunistic infections. Pharmacogenomics is the use of molecular-based tests to predict the response to specific therapies and to monitor the response of the disease to the agents administered. The best examples of pharmacogenomics in infectious diseases are the use of viral load and resistance genotyping to select and monitor antiviral therapy of AIDS and chronic hepatitis. This application improves disease outcome; shortens length of hospital stay; reduces adverse events and toxicity; and facilitates cost-effective therapy by avoiding unnecessary expensive drugs, optimizing doses and timing, and eliminating ineffective drugs. Molecular strain typing of microorganisms is now well recognized as an essential component of a comprehensive infection control program that also involves the infection control department, the infectious disease division, and pharmacy. Molecular techniques for establishing presence or absence of clonality are effective in tracking the spread of nosocomial infections and streamlining the activities of the infection control program. A comprehensive infection control program uses

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active surveillance by both infection control practitioners and the clinical microbiology laboratory to identify clusters of infections with a common microbial phenotype (same species and antimicrobial susceptibility profile). The isolates are then characterized in the laboratory by using one of a number of molecular typing methods to confirm or refute clonality. 19.3.6.3 Challenges and Opportunities The field of molecular infectious disease testing has grown so rapidly that the diagnostic industry has not kept up. To fill this void, independent investigators have turned to the development of user-defined, or “homebrew,” molecular detection methods in the clinical laboratory. The implementation of user-defined NAA testing has revolutionized clinical molecular infectious disease testing. In addition, commercially-available non-FDA-approved NAA assays are increasingly becoming available as analyte specific reagents (ASRs). Though all reagents necessary for the amplification reaction can be purchased commercially, assay development and verification studies must be performed by individual laboratories. In many cases, there are no comparative studies between user-defined NAA procedures, including ASRs, limiting the comparative value of assays between institutions (particularly in viral load monitoring) and restricting the application of such procedures to more experienced laboratories. It is not without considerable cost that a molecular infectious disease diagnostic lab is developed. It represents an institutional commitment because the costs may only be offset when analysis of hospital-wide cost savings is employed (i.e. shorter hospital stays, decreased use of unncecessary antibiotics). The costs incurred not only stem from instrumentation purchases, but also from the dedicated, expert staff required for such testing. Since many academic medical centers have resorted to implementing user-defined assays, verification and validation studies are substantial and require extensive resources, including time, staff, and expertise. These studies are crucial to defining the performance of the assay and determining appropriate clinical utilization. The FDA, diagnostic companies, and major molecular infectious disease laboratories need to work together to resolve the poor standardization that exists between laboratories using user-defined assays or ASRs.

19.4 Conclusion The applications of molecular technology in clinical microbiology are endless, but challenges also abound. We are still learning what many NAA results mean in terms of infectious etiology. With the use of molecular technology to detect potential etiologic agents of disease, we need to remember Koch’s postulates. Is the mere presence of an organism’s nucleic acid convincing evidence of disease causation? Undoubtedly, additional clinical scientific evidence is needed to make such a claim, and such evidence or lack thereof should be considered when interpreting molecular infectious disease results. Though there is still much to be learned regarding

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the appropriate application and interpretation of molecular infectious disease testing, there are numerous exciting opportunities on the horizon. User-defined assays and ASRs have allowed experienced laboratories to offer critical diagnostic services that have yet to become available with FDA clearance. As investigators refine molecular applications for infectious disease testing, diagnostic companies market such applications, quality control and government organizations standardize results, and as costs associated with implementation decrease and reimbursement increases, molecular infectious disease testing will not only be available in academic medical centers and reference laboratories, but will also transition to community hospitals, thus more globally impacting patient care.

References Ameen, M., Arenas, R., 2008, Emerging therapeutic regimes for the management of mycetomas. Expert Opin Pharmacother 9(12):2077–2085. Badiee, P., Kordbacheh, P., Alborzi, A., Malekhoseini, S., Ramzi, M., Mirhendi, H., Mahmoodi, M., Shakiba, E., 2009, Study on invasive fungal infections in immuno compromised patients to present a suitable early diagnostic procedure. Int J Infect Dis 13(1):97–102. Bisen, P.S., Garg, S.K., Tiwari, R.P., Tagore, P.R., Chandra, R., Karnik, R., Thaker, N., Desai, N., Ghosh, P.K., Fraziano, M., Colizzi, V., 2003, Analysis of shotgun expression library of Mycobacterium tuberculosis genome for immunodominant polypeptides: Potential use in serodiagnosis. Clin Diagn Lab Immunol (Am Soc Microbiol, Washington) 10(6):1051–1058. Boyadzhyan, B., Yashina, T., Yatabe, J.H., Patnaik, M., Hill, C.S., 2004, Comparison of the APTIMA CT and GC assays with the APTIMA combo 2 assay, the Abbott LCx assay, and direct fluorescent-antibody and culture assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol 42(7):3089–3093. Brechot, C., 1993, Polymerase chain reaction for the diagnosis of viral hepatitis B and C. Gut 34:39–44. Cinque, P., Vago, L., Terreni, M.R., Brytting, M., Marenzi, R., Castagna, A., Lazzarin, A., Linde, A., 1995, Diagnosis of cytomegalovirus infection of the nervous system in AIDS by polymerase chain reaction analysis of cerebrospinal fluid. Scand J Infect Dis Suppl 1995(99):92–94. Ciotti, M., Sesti, F., Paba, P., Benedetto, A., Patrizi, L., Criscuolo, A., Piccione, E., Branca, M., Syrjänen, K., Favalli, C., 2004, Human papillomavirus (HPV) testing in the management of women with abnormal Pap smears. Experience of a colposcopy referral clinic. Eur J Gynaecol Oncol 25(5):577–584. DePietropaolo, D.L., Powers, J.H., Gill, J.M., Foy, A.J., 2006, Diagnosis of Lyme disease. Del Med J 78(1):11–18. Fardy, J.M., 2009, Evaluation of diagnostic tests. Methods Mol Biol 473:127–136. Faye, O., Faye, O., Dupressoir, A., Weidmann, M., Ndiaye, M., Alpha Sall, A., 2008, One-step RT-PCR for detection of Zika virus. J Clin Virol 43(1):96–101. Garland, S.M., Tabrizi, S.N., 2004, Diagnosis of sexually transmitted infections (STI) using selfcollected non-invasive specimens. Sex Health 1(2):121–126. Ichhpujani, R.L., Bhatia, R., 1984, Immunodiagnosis of bacterial meningitis. J Commun Dis 16(1):30–37. Jeffery, K.J., Read, S.J., Peto, T.E., Mayon-White, R.T., Bangham, C.R., 1997, Diagnosis of viral infections of the central nervous system: clinical interpretation of PCR results. Lancet 349(9048):313–317. Jeffrey, M., Martin, S., González, L., Ryder, S.J., Bellworthy, S.J., Jackman, R., 2001, Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep. J Comp Pathol 125(4):271–284.

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Lebech, A.M., 2002, Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections and studies on taxonomic classification. APMIS Suppl 105:1–40. Mori, T., Sakatani, M., Yamagishi, F., Takashima, T., Kawabe, Y., Nagao, K., Shigeto, E., Harada, N., Mitarai, S., Okada, M., Suzuki, K., Inoue, Y., Tsuyuguchi, K., Sasaki, Y., Mazurek, G.H., Tsuyuguchi, I., 2004, Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens. Am J Respir Crit Care Med 170(1):59–64. Morinet, F., 1991, Virologic diagnosis of papillomavirus infections. Rev Prat Feb 41(4): 327–328. Msuya, S.E., Uriyo, J., Hussain, A., Mbizvo, E.M., Jeansson, S., Sam, N.E., Stray-Pedersen, B., 2009, Prevalence of sexually transmitted infections among pregnant women with known HIV status in northern Tanzania. Reproductive Health 6:4. O’Farrell, N., Morison, L., Moodley, P., Pillay, K., Vanmali, T., Quigley, M., Sturm, A.W., 2008, Genital ulcers and concomitant complaints in men attending a sexually transmitted infections clinic: implications for sexually transmitted infections management. Sex Transm Dis 35(6):545–549. Orle, K.A., Gates, C.A., Martin, D.H., Body, B.A., Weiss, J.B., 1996, Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers. J Clin Microbiol 34(1):49–54. Paternoster, D.M., Cester, M., Resente, C., Pascoli, I., Nanhorngue, K., Marchini, F., Boccagni, P., Cillo, U., Ribaldone, R., Amoruso, E., Cocca, N., Cuccolo, V., Bertolino, M., Surico, N., Stratta, P., 2008, Human papilloma virus infection and cervical intraepithelial neoplasia in transplanted patients. Transplant Proc 40(6):1877–1880. Pawlotsky, J.M., 2002, Molecular diagnosis of viral hepatitis. Gastroenterology 122(6):1554–1568. Pflaller, M., Herwaldt, H., 1997, Role of molecular epidemiology in infection control. Infect Dis Clin North Am 11(2):257–278. Schachter, J., Chernesky, M.A., Willis, D.E., Fine, P.M., Martin, D.H., Fuller, D., Jordan, J.A., Janda, W., Hook, E.W. 3rd, 2005, Vaginal swabs are the specimens of choice when screening for Chlamydia trachomatis and Neisseria gonorrhoeae: results from a multicenter evaluation of the APTIMA assays for both infections. Sex Transm Dis 32(12):725–728. Schluger, N.W., Kinney, D., Harkin, T.J., Rom, W.N., 1994, Clinical utility of the polymerase chain reaction in the diagnosis of infections due to Mycobacterium tuberculosis. Chest 105: 1116–1121. Silva, L.K., Silva, M.B.S., Rodart, I.F., Lopes, G.B., Costa F.Q., Melo, M.E., Gusmão, E., Reis, M.G., 2006, Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil. Braz J Med Biol Res 39(5): 595–602. Tang, Y.W., Procop, G.W., Persing, D.H., 1997, Molecular diagnostics of infectious diseases. Clin Chem 43:2021–2038. Tiwari, R.P., Tiwari, D., Garg, S.K., Chandra, R., Bisen, P.S. 2005, Glycolipids of mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis. Clin Diagn Lab Immunol (Am Soc Microbiol, Washington) 12:465–473. Tiwari R.P., Bharmal, R.N., Kartikeyan, S., Bisen, P.S., 2007, Development of a rapid liposomal agglutination card test for the detection of glycolipid antigen in patients with meningial, pulmonary and other extra-pulmonary tuberculosis. Int J Tuberculosis Lung Dis (International Union Against Tuberculosis and Lung Disease, Paris, France) 11(10):1143–1151. Tiwari, R.P., Hattikudur, N.S., Bharmal, R.N., Kartikeyan, S., Deshmukh, N.N., Bisen, P.S., 2007, Modern approaches to a rapid diagnosis of tuberculosis: Promises and challenges. Tuberculosis (Elsevier The Netherland) 87:193–201. Vázquez, F., Lepe, J.A., Otero, L., Blanco, M.A., Aznar, J., 2008, Microbiological diagnosis of sexually-transmitted infection. Enferm Infecc Microbiol Clin 1:32–37. White, M.J., Sadalla, J.K., Springer, S.R., Counselman, F.L., 2005, Is the presence of Trichomonas vaginalis a reliable predictor of coinfection with Chlamydia trachomatis and/or Neisseria gonorrhoeae in female ED patients? Am J Emerg Med 23(2):127–130.

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Chapter 20

Diagnosis of Mutation and Genetic Disorders

Abstract All diseases have a genetic component. However, the extent to which genes contribute to disease varies and much remains to be learned. Mutations may be inherited or developed in response to environmental stresses such as viruses or toxins. The ultimate goal is to use this information to treat, cure, or if possible, prevent the development of disease. Advances in understanding the genetic mechanisms behind these diseases enable the development of early diagnostic tests, new treatments, or interventions to prevent disease onset or minimize disease severity. Keywords Single gene disorders · Mutations · Genetic disorders · DNA diagnosis · Cystic fibrosis transmembrane conductance regulator gene · CFTR · BRCA1 and BRCA2 · Retinoblastoma · Inherited disorder · Monogenic disorders · Pharmacogenetics · Duchenne muscular dystrophy · Haemophilia A and B · Phenylketonuria · Thalassaemia · Multiple endocrine neoplasia · Factor V · Hemochomatosis · Colorectal cancer · Comparative genome hybridization

20.1 Prologue The exponential increase in the discovery of genes over the past few years has transformed the DNA diagnosis of genetic disorders from a minor research-based activity to a major professional operation. For any genetic disease, once the defective gene is identified, knowledge of the pathogenic mutations is indispensable to offer DNA diagnosis. DNA diagnosis is offered at pre- and postnatal levels either by direct or indirect approaches. Direct mutational analysis and linkage studies with highly polymorphic intragenic markers are carried out depending on the feasibility of their detection. This is not as easy as it may sound, because many genes, including the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, the breast cancer genes (BRCA1 and BRCA2), and the retinoblastoma gene (RBI), lack mutational hot spots necessitating an exhaustive analysis of coding and flanking intronic and regulatory sequences. M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_20,

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20.2 Concept The analysis of human DNA, RNA, chromosomes, proteins and certain metabolites in order to detect heritable disease-related genotypes, mutations, phenotypes or karyotypes for clinical purposes is essential. In the past decade, with the availability of technology and knowledge fueled by the investment and interest in the Human Genome Project, molecular diagnostics has recently enabled laboratories to offer diagnostic and predictive tests for inherited disorders (Harper et al., 2008). The technology will continue to become easier to apply and more affordable. Large amounts of genetic information can be determined at increasing efficiencies. Historically, genetic testing focused on single-gene disorders, where a disease is caused by a mutation in one gene. The classic example is sickle cell anemia. A single nucleic acid base change is responsible for the sickle cell trait, when inherited from one parent or sickle cell disease when the nucleic acid base change is inherited from both parents. Although molecular diagnostics is available, sickle cell disease is most often diagnosed with hemoglobin electrophoresis. For single-gene disorders, mutation analysis is useful in diagnosis, confirmation of diagnosis, predisposition, prognosis and family planning. As with Huntington’s disease, the identification of the gene and availability of testing opened another door to additional social and medical issues. In contrast, testing an individual with multiple endocrine neoplasia type-2 (MEN-2) for the RET proto-oncogene can avoid medullary carcinoma if the individual is positive and undergoes a prophylactic thyroidectomy. The pace of the molecular dissection of human disease can be measured by looking at the catalog of human genes and genetic disorders identified so far in Mendelian Inheritance in Man (Holzman and Watson, 2002) and in OMIM, its online version, which is updated daily (www.ncbi.nlm.nih.gov/omim). For 1100 genes, at least one disease-related mutation has been identified. Because different mutations in the same gene often result in more or less distinct disorders, the total number of diseases for which OMIM lists mutations approaches 1500. The number of disease genes discovered so far is 1112. This number does not include at least 94 disease-related genes identified as translocation gene-fusion partners in neoplastic disorders. Apart from the genes that cause monogenic disorders, the discovery of functional sequence variants that confer genetic susceptibility to common multifactorial disorders, such as cardiovascular disease, psychiatric disorders, autoimmune disorders and cancer, also creates demand for high throughput testing for clinically relevant polymorphisms. Single Nucleotide Polymorphisms (SNPs) are the most abundant type of DNA sequence variations in the human genome, though only miniscule fraction causes significant changes in amino acid sequence. One area where SNPs may have an immediate impact on patient care is the individual response to drug therapy, commonly referred to as pharmacogenetics. A genetic variability in N-acetyl transferase (NAT-2), for instance, is associated with a high incidence of peripheral neuropathy when taking isoniazid, an antituberculosis drug. A variant in the core promoter of the ALOX5 gene, on the other hand, is responsible for

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the failure of some asthma patients to respond to treatment with ALOX5-pathway modifiers. The ultimate method for detection and definition of mutations is direct sequencing. But it comes at significant cost and labour, and the vast majority of sequencing reactions will only exclude the presence of a mutation. This has led to the development of physical, chemical and biological mutation screening methods that exclude the presence of mutations at a fraction of the cost of sequencing, but provide little, if any, information on the location and nature of sequence variation in mutated gene fragments. Most methods do well with regard to specificity, but fail miserably when it comes to sensitivity. They include such popular methods as single-strand conformation analysis, gel electrophoresis-based heteroduplex analysis, and denaturing gradient gel electrophoresis. The diagnosis of a genetic disease requires a comprehensive clinical examination composed of three major elements: 1. A physical examination 2. A detailed medical family history 3. Clinical and laboratory testing if available. While primary care providers may not always be able to make a definitive diagnosis of a genetic disease, their role is critical in collecting a detailed family history, considering the possibility of a genetic disease in the differential diagnosis, ordering testing as indicated and when available, appropriately referring patients to genetic specialists.

20.3 Factors Regulating a Genetic Disease There are several factors that raise the possibility of a genetic disease in a differential diagnosis. One major factor is the occurrence of a condition among family members that is disclosed when the family history is obtained. The occurrence of the same condition in more than one family member (particularly first-degree relatives), multiple miscarriages, stillbirths, and childhood deaths are all suggestive of a genetic disease. Additionally, family history of common adult conditions (heart disease, cancer, and dementia) that occur in two or more relatives at relatively young ages may also suggest a genetic predisposition. Other clinical symptoms that are suggestive of a genetic disease include developmental delay/mental retardation and congenital abnormalities. Dysmorphologies, often involving the heart and face, as well as growth problems are suggestive of a genetic disorder caused by an inherited mutation, a spontaneous mutation, a teratogen exposure, or unknown factors. While these clinical features may be caused by a number of factors, genetic conditions should also be considered as part of the differential diagnosis, particularly if the patient expresses several clinical features together that might be indicative of a syndrome (for example, mental retardation, distinct facies, and heart defect). Some physical features may appear unique or slightly different than the average such as wide-set or droopy eyes, flat face,

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short fingers, and tall stature. While these rare and seemingly mild features may not immediately be suggestive of a genetic disease to a primary care provider, an evaluation by a genetics specialist may be helpful in ruling in/out a genetic disease. While many genetic conditions appear during childhood, a genetic condition should not entirely be ruled out in adolescents or adults. Often a genetic disease can remain undetected for several years until an event such as puberty or pregnancy triggers the onset of symptoms or the accumulation of toxic metabolites manifests in disease. In these cases, a detailed family history and physical examination should be performed and a referral made to a genetics specialist if needed.

20.4 Genetic Testing Genetic tests can be used for many different purposes (Holtzman and Watson, 2002; Fig. 20.1). Newborn screening is the most widespread use of genetic testing. Almost every newborn in the U.S. is screened for several genetic diseases. Early detection of these diseases can lead to interventions to prevent the onset of symptoms or minimize disease severity. Carrier testing can be used to help couples to learn if they carry and thus risk passing to their children – an allele for a recessive condition such as cystic fibrosis, sickle cell anaemia, and Tay-Sachs disease. This type of testing is typically offered to individuals who have a family history of a genetic disorder and to people in ethnic groups with an increased risk of specific genetic conditions. If both parents are tested, the test can provide information about a couple’s risk of having a child with a genetic condition. Prenatal diagnostic testing is used to detect changes in a foetus’s genes or chromosomes. This type of testing is offered to couples with an increased risk of having a baby with a genetic or chromosomal disorder. A tissue sample for testing can be obtained through amniocentesis or chorionic villus sampling. Predictive or predispositional genetic testing can identify individuals at risk of getting a disease prior to the onset of symptoms. These tests are particularly useful if an individual has a family history of a specific disease and an intervention is available to prevent the onset of disease or minimize disease severity. Predictive testing can identify mutations that increase a person’s risk of developing disorders with a genetic basis, such as certain types of cancer. Several different methods are currently used in genetic testing laboratories. The type of test will depend on the type of abnormality that is being measured. In general, three major types of genetic testing are available: cytogenetic, biochemical, and molecular testing.

20.4.1 Cytogenetic Testing Cytogenetics involves the examination of whole chromosomes for abnormalities. Chromosomes of a dividing human cell can be clearly analyzed under a microscope. White blood cells, specifically T lymphocytes, are the most readily accessible cells for cytogenetic analysis since they are easily collected from blood and are capable

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Fig. 20.1 Genetic tests may be used to confirm a diagnosis in a symptomatic individual or used to monitor prognosis of a disease or response to treatment

of rapid division in cell culture. Cells from other tissues such as bone marrow (for leukemia), amniotic fluid (prenatal diagnosis), and other tissue biopsies can also be cultured for cytogenetic analysis Following several days of cell culture, chromosomes are fixed, spread on microscope slides, and then stained. The staining methods for routine analysis allow each of the chromosomes to be individually identified. The distinct bands of each chromosome revealed by staining allow for analysis of chromosome structure.

20.4.2 Biochemical Testing The enormous numbers of biochemical reactions that routinely occur in cells require different types of proteins. Several classes of proteins exist to fulfill multiple functions, such as enzymes, transporters, structural proteins, regulatory proteins, receptors, and hormones. A mutation in any type of protein can result in disease if the mutation results in failure of the protein to correctly function. Clinical testing for a biochemical disease utilizes techniques that examine the protein instead of the gene. Tests can be developed to directly measure protein activity (enzymes), level of metabolites (indirect measurement of protein activity), and the size or quantity of protein (structural proteins). These tests require a tissue sample in which the protein is present, typically blood, urine, amniotic fluid, or cerebrospinal fluid. Because proteins are more unstable than DNA and can degrade quickly, the sample must

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be collected, stored properly, and shipped promptly according to the laboratory’s specifications.

20.4.3 Molecular Testing For small DNA mutations, direct DNA testing may be the most effective method, particularly if the function of the protein is not known and a biochemical test cannot be developed. A DNA test can be performed on any tissue sample and requires very small amounts of sample. Some genetic diseases can be caused by many different mutations, making molecular testing challenging. For example, more than 1000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis (CF). It would be impractical to sequence the entire CFTR gene to identify the causative mutation since the gene is quite large. However, since the majority of CF cases are caused by approximately 30 mutations, this smaller group of mutations is first tested before more comprehensive testing is performed (Grody, 1999).

20.5 Current Status of Molecular Diagnosis of Some Common Genetic Diseases Beyond the complexity of genetic sequencing, with rigorous quality control and assurance, is the difficulty in interpreting the results. As with HIV genotyping, some mutations have no known clinical significance and others impart antiretroviral drug resistance. Sometimes, two mutations act to cancel out the impact of one another. Likewise, some mutations in the gene associated with Gaucher’s disease cause neuronopathic disease and other mutations do not. As a final example, some mutations in the CFTR cause cystic fibrosis. Other mutations have low penetrance. That is, these genes seem to have no clinical significance in the majority of individuals, but when combined with other gene expressions, impart classical or nonclassical cystic fibrosis disease. The current state of molecular diagnosis of some common genetic diseases, including cystic fibrosis, Duchenne muscular dystrophy, haemophilia A and B, phenylketonuria, and thalassaemia, has been reported. Data on carrier detection and prenatal diagnosis are presented and some objective problems and obstacles hampering efficient molecular diagnosis are discussed. The necessity for molecular diagnosis of some other inherited diseases (for example, von Willebrand’s disease, Martin-Bell syndrome, polycystic kidney disease, Huntington’s disease, and myotonic dystrophy) is stressed. The need for establishing new diagnostic centres dealing with the most common diseases, as well as rare genetic diseases, is substantiated. Perspectives on the implementation of new molecular methods and new technical approaches (preimplantation embryo diagnosis, fetal cells selected from maternal blood) are briefly outlined. It is still too early to estimate the significance of our Human Genome Programme as a whole, but its important impact on medical genetic studies in this country is quite obvious and has been highly appreciated.

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There are a myriad of technologies capable of detecting single mutations or SNPs. These platforms are capable of performing medium throughput testing with standard laboratory robotic liquid handlers and at a reasonable hardware cost. These methods include allele-specific oligonucleotide hybridization, PCR RFLP, allele-specific PCR, Line Probe Assays (reverse dot blots), Invader (Third Wave Technologies), ReadIT (Promega), Nanochips (Nanogen), Homogenous PGR (includes TaqMan and Molecular Beacon) and many others. Gene quantitation can be done by several techniques, including fluorescent in situ hybridization (FISH), usually performed in the cytogenetics laboratory with the use of a computerized fluorescent microscope, real-time PCR analysis and comparative genome hybridization.

20.5.1 Cystic Fibrosis Cystic fibrosis (CF) is one of the most commonly inherited diseases in the Caucasian populations (Bouchara et al., 2009). Those affected have high levels of sodium and chloride (salt) in their sweat. More importantly, a thick, sticky mucous in the lungs causes persistent coughing, wheezing and frequent lung infections, including pneumonia. In 1989, a research team led by Dr. Francis Collins at the University of Michigan and Lap-Chee Tsui and John Riodan at Toronto’s Hospital for Sick Children discovered the gene responsible for cystic fibrosis. The protein is the cystic fibrosis transmembrance conductance regulator (CFTR). Since the discovery of the most common mutation that causes cystic fibrosis, approximately 1000 additional mutations have been identified. Given that approximately 10 million Americans are carriers for cystic fibrosis and 30,000 have the disorder, testing for cystic fibrosis carrier status prior to conception (or if necessary, early after conception) is currently recommended. While cystic fibrosis is not curable, there are some treatments that greatly increase the life span and quality of life for patients with CF. Today, cystic fibrosis mutational analysis is the most commonly performed molecular diagnostic test for inheritable disorders. Patterns of allelic polymorphisms in CF have been analysed and studied. Large scale testing of the AF508 mutation has become possible after adopting the PCR technique for dried blood spots on filter paper. The other major mutations of the CFTR gene known to be quite common in western populations (G551D, R553X, R334W, W1282X, R551X, 1716+ 12T-∗ C) are detected only occasionally in Russian samples (1–3%). However, CFTR gene mutation 3732delA (exon 19), recently found in southern France, was detected in almost 7% of Russian CF chromosomes. Moreover, 1677delTA, originally discovered turned out to be a major mutation inthe populations of the Black Sea Basin. Three more new CFTR gene mutations (E504Q exon 10, W1282R exon 19, and S 1196X) have been recently shown by SSCP analysis followed by direct sequencing. At least three different mutations, AF508, 1677delTA, and 3732delA, might be used both for carrier detection and prenatal diagnosis of CF. Identification of new major CFTR gene mutations, specific to our native populations, as well as RFLP

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analysis of new intragenic polymorphisms, such as the recently discovered highly polymorphic minisatellite DNA sequences in introns 6, 8, and 17b, is helping in more efficient application of molecular analysis in CF patients (Grody, 1999).

20.5.2 Duchenne Muscular Dystrophy Molecular diagnosis of Duchenne muscular dystrophy (DMD) were carried out with intragenic and flanking DNA probes. They were later supplemented with multiplex polymerase chain reaction (MPCR) for exon deletion detection in the dystrophin gene. Both carrier detection and prenatal diagnosis have been done. A relatively low deletion detection rate with the standard set of exons tested by MPCR, a somewhat unusual pattern of deletion distribution along the DMD cDNA, and a significant proportion of extensive deletions extending through the major part of the gene. One of the possible approaches, not yet tried here so far, concerns RNA amplification supplemented with MPCR analysis of cDNA. Of special diagnostic value for at least some male fetuses at risk might also be the application of western blotting to muscle biopsies or direct immunocytochemical studies of dystrophin in muscle fibres. This approach might be of great benefit for the otherwise uninformative DMD families requesting prenatal diagnosis during the second trimester of pregnancy.

20.5.3 Haemophilia A Molecular diagnosis of haemophilia A was carried out using DNA probes. Highly polymorphic flanking DNA (probe StI4/TaqI) and intragenic polymorphic sites HindIII (intron 19), BclI (intron 18), and XbaI (intron 22) were used both for population studies and diagnostic purposes. Some intragenic polymorphic sites were used for polymerase chain reaction detection. Several prenatal diagnoses by PCR have been carried out. Mutation identification by SSCP analysis followed by direct sequencing of altered exons and amplification-mismatch detection studies of the factor VIII gene are now in progress.

20.5.4 Haemophilia B Intragenic DNA probes are available for RFLP analysis of the factor IX gene. Southern blot RFLP analysis with these probes was later replaced by PCR for detection of intragenic polymorphic sites TaqI, XmnI (both in intron C), and HinfI/DdeI (intron A). Polymorphism identification was done with original sets of oligoprimers. Thus, direct identification of mutations by means of SSCP analysis or by the amplification-mismatch detection technique is advisable.

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20.5.5 Phenylketonuria Large scale newborn screening programmes for phenylketonuria (PKU), either by the Guthrie test or by an automated fluorescent assay are in practice. According to already available data the frequency of PKU in newborns varies between 1 in 5000 to 1 in 8000 in different regions, with an average of around 1 in 6000. Molecular analysis of PKU was initiated with the cDNA probe of the PAH gene. These studies were substantially helped later by the PCR method. Allele specific hybridisation supplemented with direct sequencing of exon 12 of the PAH gene showed the mutation in codon 408 as well as the exon-intron splicing mutation.

20.5.6 β Thalassaemia Molecular studies of β thalassaemia was started in 1975 and was initially confined to mRNA analysis and its application to deletion detection in the A globin gene (L Lymborskaya). RFLP analysis in 32 thalassaemia patients with a severe form of the disease showed 11 different haplotypes, one of which was encountered in half of all affected subjects. This particular haplotype was actually in strong linkage disequilibrium with mutations in codon 8. One of these mutations (deletion of a G nucleotide between codons 82 and 83) was found for the first time. Oligoprimer sets suitable for allele specific amplification and thus for direct identification of globin gene mutations have been suggested.

20.5.7 Wilson’s Disease (Hepatolenticular Degeneration) Identification and biochemical investigations of copper binding protein caeruloplasmin (CP) in Wilson’s disease patients were later extended to CP mRNA and CP gene molecular analysis. However, these studies failed to disclose any mutations of the CP gene in patients and thus indicated that the CP gene is not involved by itself in Wilson’s disease. Curiously enough, in situ gene mapping with an original fragment of rat CP-DNA as a hybridisation probe disclosed a positive hybridisation signal not only on chromosome 3 (3q23–25, close to the transferrin gene) but also on chromosome 13 (13q23–24), that is, somewhere very close to the still unknown gene responsible for Wilson’s disease. Thus the problem of the molecular nature of Wilson’s disease is still awaiting a solution. Meanwhile chromosome 13 is one of the genome units selected by our Human Genome Project for detailed molecular analysis. Identification of the gene for Wilson’s disease is one of the urgent tasks.

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20.5.8 α Antitrypsin Deficiency DNA analysis of α antitrypsin deficiency (AD) was carried out. RFLP analysis was carried out in 659 patients with chronic non-specific lung disease and a substantial preponderance of the abnormal Z allele was found. An unusual neutral mutation of the MaeIII site in exon 3 of the AT gene in 20–30% of normal subjects with the MI allele, as well as its linkage disequilibrium with the Z allele in AD patients, was discovered. An allele specific amplification system for direct detection of the common mutation in codon 342 of AD patients with the Z haplotype was elaborated and reported.

20.5.9 Familial Hypercholesterolaemia and Other Lipoprotein Disorders Molecular analysis has been confined to 20 families with familial hypercholesterolemia. No major mutations or rearrangements in the low density lipoprotein receptor gene have been reported so far. RFLP analysis of different polymorphic sites of the receptor gene in normal and affected subjects is in progress. Correlation of particular genotypes of Apo CIII, Apo B 100, and Apo Al genes with blood cholesterol lipoprotein levels in patients with cardiac ischaemic disease and in the general population was reported. A simple approach based on PCR mediated site directed mutagenesis has been suggested for the identification of the common mutation in codon 3500 of the apo Bio gene.

20.5.10 Huntington’s Disease Gene Dr. George Huntington, along with his father and grandfather, published their observations in 1872 on familial cases of chorea near their home on Long Island, NY. The genetic disorder they described is now known as “Huntington’s disease” (HD). Nearly 1% of Americans has HD or is at risk of passing along the disease to a child. HD affects as many people as hemophilia, cystic fibrosis or muscular dystrophy combined. In 1993, the HD gene was isolated and, eventually, a direct genetic test was developed that can accurately determine carrier status for the HD gene. The HD gene was found to contain a specific section with a pattern of so-called “trinucleotide repeats” which is expanded in people with HD. In most cases, the repeated pattern occurs 30 times or less. In HD it occurs more than 40 times (Harper et al., 2008). The test cannot predict when symptoms will begin, and therapy is palliative. In the absence of effective treatment and a cure, most individuals at risk elect not to take the test. HD is one of many trinucleotide expansion diseases characterized by genetic anticipation. This phenomenon manifests when a genetic disease either presents earlier, or presents with more severe symptoms in subsequent generations. For example, myotonic dystrophy has a broad spectrum of presentation varying from congenital myotonia to the seventh decade of life. Because of genetic anticipation,

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early and accurate diagnosis is vital for parents who may have minimal or late onset symptoms but can have children who are more severely affected. The advent of pre-implantation genetics allows for the detection of embryos that are homozygous for inheritable recessive disorders. Through detection of a mutation of the dystrophin gene in a fertilized cell, a couple can avoid bringing to term a child with Duchenne’s muscular dystrophy. Approximately 30 other diseases like Huntington’s disease, TaySachs, cystic fibrosis and familial dysautonomia can be diagnosed on an embryo prior to implantation in the womb.

20.5.11 Multiple Endocrine Neoplasia Multiple endocrine neoplasia, type 2 (MEN 2) presents as two syndromes, 2A and 2B. In MEN 2A, medullary thyroid carcinoma (MTC) involving the thyroid interstitial C-cells is ultimately found in some 90–95% of affected individuals, pheochromocytoma in 25–50% and hyperparathyroidism in 15–20%. MEN 2B is characterized by MTC, pheochromocytoma, mucosal neuromata and marfanoid habitus. Classical linkage studies initiated in the early 1980s led to mapping of the MEN 2 gene to a centromeric chromosome 10 locus in 1987. Mutations of the RET protooncogene causative for MEN 2 were identified in 1993. Clinical laboratory testing became available a few years later. (Hoff et al., 2001). The discovery of molecular abnormalities of the RET proto-oncogene in MEN 2 and FMTC has significantly improved clinical management of these disorders. The identified mutations are responsible for 90–95% of all hereditary MTC. Six to seven percent of patients with apparent sporadic MTC have been found to have germline mutations of RET indicative of hereditary MTC. Application of these tests in the management of MTC has improved diagnostic accuracy, lessened the likelihood that hereditary disease will be missed in the context of apparent sporadic MTC and improved the clinical management of this disease. Many of the common disorders that plague humans are multifactorial gene disorders, including cardiovascular disease, diabetes and most types of cancer. Some genes associated with these disorders may indicate increased predisposition, just as total cholesterol and LDL cholesterol serve as important but not exclusive predictors of cardiovascular disease. With additional studies, more genes are being identified with behaviors. These genes may become more important in the future as we develop a fuller understanding of the impact of genetic factors on behavioral disorders and characteristics. Common disorders for which intensive research is underway include cardiovascular disease, diabetes, cancer, neurological diseases (including Alzheimer’s), bipolar disease, osteoporosis and behavioral disorders. The diagnostic tests that will be developed will complement current and future tests, including those involving proteomics and metabolic analysis. Many of these diagnostic tests may not live up to initial expectations and others may exceed them. Academic centers and large reference laboratories will be on the forefront of this frontier. The prospectors and

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pioneers will be followed by a growing group of laboratories as molecular diagnostic tests are more widely accepted and adopted. This is the same pattern that was observed with testing for HIV.

20.5.12 Factor V (Leiden) Mutation The factor V (Leiden) mutation (1691G > A) occurs primarily in the Caucasian population and is a major risk factor for venous thrombosis (a lifetime risk of 12–30% in affected individuals) and a lesser risk factor for arterial thrombosis (cardiovascular disease). Additionally, factor V (Leiden) mutation is associated with arterial thrombosis (especially in smokers), complications of pregnancy (including fetal loss) and increased levels of factor VIII. The factor V (Leiden) mutation leads to the laboratory finding of activated protein C resistance (APCR) and a sevenfold increase in venous thromboembolic events in heterozygous individuals and an eightyfold increase in homozygous subjects. Due to a synergistic increase in venous thrombosis risk, individuals heterozygous for the factor V mutation are at greater risk when taking oral contraceptives. When a heterozygous mutation is coupled with oral contraceptive use, risk increases synergistically to thirtyfold (Grody et al., 2001). Since laboratory tests for APCR are highly sensitive, specific and simpler to perform, APCR is usually the test of choice; however, factor V mutation analysis is recommended to confirm positive APCR tests. It is also recommended in place of APCR for patients with lupus anticoagulant, since such patients often have a falsepositive APCR test. Although this test is highly specific, identification of a mutation may occur in the absence of APCR in rare cases. Sensitivity of this test for APCR is 94%; thus, a negative result does not rule out APCR or an increased risk of venous thrombosis. More than half of thromboembolic events associated with factor V (Leiden) mutation occur in the presence of additional risk factors, such as surgery and use of oral contraceptives. Thus, factor V (Leiden) mutation is a risk factor and not an indication of thromboembolic disease.

20.5.13 Hemochromatosis Gene, HFE Hemochomatosis is an excess accumulation of iron that causes damage to organs, leading to such diseases as cirrhosis, cardiomyopathy, diabetes and arthritis. Two mutations in the HFE gene – C282Y and H63D – are associated with hemochromatosis (Hanson et al., 2001). Disease develops in less than 1% of individuals with these genotypes. These mutations have low penetrance. Other factors, such as diet, hepatotoxins and likely other genes, are important factors that lead to hemochromatosis. The role of testing may be limited to family members of individuals with hemochromatosis. Even for these individuals, biochemical testing remains the cornerstone for diagnosis.

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Many molecular diagnostic tests may be similar to the HFE gene tests that have a limited role and must be interpreted in the context of family, clinical history, and other risk factors and laboratory tests.

20.5.14 Colon Cancer Gene, APC Dr. Bert Vogelstein generated enormous excitement 15 years ago when he and his colleagues at Johns Hopkins described a series of genetic alterations leading to colorectal cancer (Vogelstein et al., 1998), that occurs in the different phases of cancer development, starting from normal epithelium and moving through adenomatous polyps to cancer. This suggested a genetic pathway in tumor development. Unfortunately, the number and nature of the genetic alterations may vary in a population. There may be a spectrum of routes that describes the genetic alterations leading to cancer. The interaction with environmental factors, including diet and other genetic factors, is open for exploration. Today, genetic testing for colorectal cancer is quite limited.

20.6 Microarray Analysis for Detection of Complex Pattern of Genes Comparative genome hybridization technique used in microarrays hold enormous hope in the field of oncology. Many solid and hematologic malignancies have gene duplications and/or gene deletions associated with them. Amplification of the protooncogene Her2/neu has been demonstrated to correlate with a higher grade of malignancy and with tumor response to the chemotherapeutic agent Herceptin. Comparative genome hybridization microarrays have the ability to scan the entire genome for such insertions and deletions and may revolutionize drug development and prognostic testing. Multiple SNP analysis is currently necessary for cystic fibrosis carrier detection, which requires 25 mutations and six polymorphisms to be analyzed simultaneously. A low-density microarray has been developed for this purpose. Suggestions have been made to screen patients for multiple drug sensitivity pharmacogenetic SNPs using a single chip. This approach suffers from HIPAA and compliance issues, as genetic testing would be performed without a definite indication. Microarrays will likely be used in one of four ways: expression arrays, resequencing microarrays, multiple genotyping arrays and comparative genome hybridization arrays. Expression arrays consist of DNA probes immobilized on chips that are used to detect mRNA, primarily in solid tumors. Promising data suggests these expression arrays can be used to predict malignant potential in stage 1 breast cancer and other malignancies. Resequencing microarrays are being developed to replace expensive DNA sequencing assays for large genes, such as BRCA1 and BRCA2. These chips may reduce the cost of BRCA1 sequencing. If this can be accomplished, the indications for BRCA1 and BRCA2 testing may be broadened.

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Genetic testing, as all clinical laboratory testing, must always be interpreted in the light of clinical findings. Molecular testing for hemochromatosis, alone, is not capable of making the diagnosis of hemochromatosis. Less than 5% of patients homozygous for C282Y in the HFe gene will ever develop symptoms of hemochromatosis. Molecular genotyping tests are extremely sensitive and specific for the mutations they are designed to detect, but may be less so for disorders associated with those diseases. These complex tests may need to be interpreted with additional clinical and routine test results. Regulatory oversight and ethical debate will strive to keep pace with the rapid advances and applications.

20.7 Conclusion Molecular diagnosis of inherited diseases will expand rapidly into clinical labs in the coming years. Laboratorians must develop internal capabilities to perform and interpret selective tests that are appropriate for their setting, understand the clinical application of these tests, examine the technical issues with performing these tests and maintain the expertise to interpret the test results in the full context of the patient and the family being tested. The future will certainly include more demand for information that will improve the lives of individuals, including testing of embryo and foetus. Technology will make it easier for laboratories to perform molecular diagnostics of common disorders. The application of microarray will aid in the discovery of complex patterns of genes, whose functions may not be understood, that define prognostic patterns and lead to therapeutic recommendations. From Mendel’s rudimentary study of peapods a century and a half ago, to the sophisticated clinical laboratories of today, the rapid growth of molecular genetics provides unsurpassed diagnostic insights into complicated hereditary diseases that affect the lives of human beings worldwide. From these insights, medical science can contribute solutions to the families who struggle to resolve hereditary issues. After all, life should be as simple as “she has your smile and my eyes.”

References Bouchara, J.P., Hsieh, H.Y., Croquefer, S., Barton, R., Marchais, V., Pihet, M., Chang, T.C., 2009, Development of an oligonucleotide array for direct detection of fungi in sputum samples from patients with cystic fibrosis. J Clin Microbiol 47(1):142–152. Grody, W.W., 1999, Cystic fibrosis: molecular diagnosis, population screening and public policy. Arch Pathol Lab Med 123:1041–1046. Grody, W.W., Griffin, J.H., Taylor, A.K., Korf, B.R., Heit, J.A., ACMG Factor V. Leiden Working Group, 2001, American College of Medical Genetics consensus statement on factor V Leiden mutation testing. Genet Med 3(2):139–148. Hanson, E.H., Imparatore, G., Burke, W., 2001, HFE gene and hereditary hemochromatosis: a HuGE review. Am J Epidemiol 154:193–206. Harper, P.S., Lim, C., Craufurd, D., 2008, Ten years of presymptomatic testing for Huntington’s disease: the experience of the UK Huntington’s Disease Prediction Consortium. J Med Genet 37:567–571.

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Hoff, A.O., Cote, G.J., Gagal, R.F., 2001, Multiple endocrine neoplasias. Annu Rev Physiol 62:377–411. Holtzman, N.A., Watson, M.S., 2002, Promoting safe and effective genetic testing in the United States: Final Report of the Task Force on Genetic Testing. Baltimore Burke, W. Genetic Testing. N Engl J Med 347:1867–1875. Vogelstein, B., Fearon, E.R., Hamilton, S.R., Kern, S.E., Preisinger, A.C., Leppert, M., Nakamura, Y., White, R., Smits, A.M., Bos, J.L., 1998, Genetic alterations daring colorectal-tumor development. N Engl J Med 319:525–532.

Chapter 21

Diagnosis of Complex Diseases

Abstract Although many types of diagnostic and carrier testing for complex disorders have been available for decades, the use of molecular methods is a relatively recent phenomenon. Such testing has expanded the range of disorders that can be diagnosed and has enhanced the ability of clinicians to provide accurate prognostic information and formulate appropriate health supervision measures. However, the proper application of these tests may be difficult because of their scientific complexity and the potential for negative, sometimes unexpected, consequences for many patients. Keywords Complex diseases · SNP · Alzheimers · Cancer · Haemophilia · Multiple sclerosis · Obesity · Diabetes · Diagnosis · Complex diseases · X-linked inheritance · Multifactorial disorders · Monogenic disorders · Marfan syndrome · Quantitative trait locus · QTL · Tumor markers

21.1 Prologue Molecular Diagnostics is constantly translating new discoveries and novel technologies into useful clinical tests that provide a molecular fingerprint of tumours and that are predictive of the response to specific therapies. We have entered a new era in medical research. Today’s technologies permit us to survey a sufficient number of letters throughout the human genome to provide a clearer picture of how life works and ultimately allow better clinical management of diseases. The most common diseases are the toughest to crack. Heart disease, cancer, diabetes, psychiatric illness, all of these are “complex” or “multifactorial” diseases, meaning that they cannot be ascribed to mutations in a single gene or to a single environmental factor. Rather they arise from the combined action of many genes, environmental factors, and riskconferring behaviors. One of the greatest challenges facing biomedical researchers today is to sort out how these contributing factors interact in a way that translates into effective strategies for disease diagnosis, prevention, and therapy. Advances in the Human Genome Project have profoundly influenced not only the search for new genes, but also the molecular diagnosis of genetic disorders (Vilain, 1998). M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_21,

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21.2 Concept Beginning in 1986, map-based gene discovery (positional cloning) became the leading method for elucidating the molecular basis of genetic diseases. Almost all medical specialties have used this approach to identify the genetic causes of some of the most puzzling human disorders. Positional cloning has also been used reasonably successfully in the study of common diseases with multiple causes (socalled complex disorders), such as type I diabetes mellitus and asthma. With the availability of the human genome sequence and those of an increasing number of other species, sequence-based gene discovery is complementing and will eventually replace map-based gene discovery. Complex diseases have complex phenotypes, and proper diagnosis requires that the analysis take into account the patient’s history and exposure to environmental factors, as well as genetic information. Signaling information is one aspect of a grandeur “biomedical informatics” approach advocated for a better understanding of a patient’s medically relevant disease phenotype. Complex diseases are genetic diseases that do not obey the single-gene dominant or single-gene recessive Mendelian law. The term complex trait is also used for phenotypes that may not be considered as diseases. The etiology of most common diseases is multifactorial, but some Mendelian conditions have a high prevalence (e.g., hereditary hemochromatosis and the genetic predisposition toward breast or colorectal cancer). The same holds true for chromosomal anomalies. The diagnosis of the complex diseases is a very difficult process and it is a very tough job in the development of the diagnostic techniques for them. The major diagnostics applied today deal mostly with the physical appearances of these diseases, but slowly the application of the modern techniques like the use of DNA analysis, cytogenetics and other molecular diagnostics are picking up the pace. These techniques have got immense potential to boost the early diagnosis of the complex diseases and in the designing of the treatment for them. The complex diseases comprise of diabetes, cancer, obesity, Alzheimer, schizophrenia, hemophilia, multiple sclerosis etc. All these diseases have been shown to be linked to one or more than one genes and have been found to be passed from one generation to the other (inheritance). The example of the X-linked inheritance is hemophilia which is passed from mother to her children, in this the son would be the patient and the daughter would be carrier, both having the probability of 50% each. The exponential increase in the discovery of genes over the past few years has transformed the DNA diagnosis of genetic disorders from a minor research-based activity to a major professional operation. For any genetic disease, once the defective gene is identified, knowledge of the pathogenic mutations is indispensable to offer DNA diagnosis. DNA diagnosis is offered at pre- and postnatal levels either by direct or indirect approaches. Direct mutational analysis and linkage studies with highly polymorphic intragenic markers are carried out depending on the feasibility of their detection. Apart from the genes that cause monogenic disorders, the discovery of functional sequence variants that confer genetic susceptibility to common multifactorial disorders, such as cardiovascular disease, psychiatric disorders,

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autoimmune disorders and cancer, also creates demand for high throughput testing for clinically relevant polymorphisms. An SNP involves any nucleotide of the human genome in which two different bases can be found in the population. SNPs are the most common type of human genetic variation. Because they have only two alleles, they are less informative than microsatellites. This drawback is overcome by their high density throughout the genome and the simplicity and possible automation of their analysis. They may become one of the most powerful tools in human genetics for identifying disease genes and mapping complex traits by linkage. As larger portions of the human genome are sequenced, one might speculate that mapping tools would become obsolete for diagnostic studies. Direct mutation detection is considered the gold standard of molecular diagnosis because all of the methods based on linkage of polymorphic markers may be impeded by a lack of informative markers or by misinterpretations resulting from recombination events, new mutations, or misattribution of paternity. SNPs will most likely be used widely for the localization of loci for complex traits. It remains to be seen whether SNPs will also be major tools for molecular diagnosis. One of their unique characteristics is the possibility of a relatively simple automation of their analysis, which may lead in the future to molecular screening of large populations. Because of their potential for high throughput, SNPs will have increasing application for predisposition traits when linkage disequilibrium is demonstrated with disease genes.

21.3 Background Information It is estimated that anywhere from 50,000 to 100,000 genes are contained in the 46 chromosomes present in each human cell. A genetic locus is the place on homologous chromosome pairs where genes are located. Each gene is composed of 2 alternative copies known as alleles, one originating from the maternally derived chromosome and the other originating from the paternally derived chromosome of each chromosome pair. Genes are composed of DNA, and the products of genes are most often proteins that may be used for a variety of purposes, including structural development, regulation of cellular function, enzyme activity, and control of metabolic pathways. Although most changes in the DNA base-pair composition of genes do not result in disease and are known as polymorphisms, some gene changes alter gene function to such a degree that clinical disease is manifested, and these are known as mutations. Mostly genetic disease is caused by single base-pair deletions, additions, or substitutions. However, some disorders are caused by large-scale gene abnormalities, such as deletions of the entire gene that can be detected by newer methods of diagnosis, such as molecular cytogenetic analysis. As in all diagnostic testing, it is most important that the clinician have a reasonable index of suspicion based on clinical signs and symptoms that suggest a specific diagnosis.

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Advances in genetics are likely to uncover new genes involved in complex diseases such as diabetes, hypertension, cancer, Alzheimer’s disease, and others (Mouulsky, 2006). This may increase the demand for genetic testing to predict individual predisposition to future illness in individuals with low-to-average risk and provide opportunities for preventive measures and early intervention. The advances in genetics and the constant changes in gene testing technologies present special challenges to health professionals. There is a need for better education of health professionals about the genetic testing process to improve patient care.

21.4 Detecting New Metabolic Disease Pathways The majority of publications reporting genetic studies of complex diseases investigate candidate genes and known metabolic pathways. The major problem with any strategy for analyzing a candidate gene or metabolic pathway is that we look wherever we can (that is, among the candidates that we already know) and we most probably overlook other essential genes or pathways because of our ignorance of human biology. Proteins, the products of genes, perform their functions by interacting with each other in coordinated networks. But, only a fraction of these networks have been identified and characterized through classical biochemistry, structural analyses, and assays of activity. A much more comprehensive view of how proteins interact with each other inside cells will become possible with sequence information from multiple species, including humans. Knowing the full complement of our genes, we should be able to identify all of the metabolic pathways in the human body, no matter how short the half-life of the participating proteins or how small the developmental window during which the pathway is turned on.

21.5 Phenotypic Variations in Simplex Diseases There are major problems associated with dissecting the molecular basis of even simple monogenic diseases caused by mutations in a single gene. Principal among these are the modifying effects of other genes. No gene operates in a vacuum; rather, each gene busily interacts either directly or through its protein product with many other genes and gene products. This results in marked variations in the symptoms of patients with the same disease. Of the 1500 or so monogenic diseases for which the mutated gene has been identified, there are only a few where the effects of other genes on disease pathogenesis have been studied. Existing information about monogenic diseases, such as cystic fibrosis and Hirschsprung disease, demonstrates that certain modifier genes cause variations in the clinical phenotype of these disorders. It is probable that many diseases that are considered monogenic will turn out to be “complex” disorders. Such complexity may be attributable to the typically unpredictable effects of gene mutations on the encoded protein and on the metabolic pathway in which the protein acts. As exemplified by Marfan syndrome, there could be an expression threshold below which the mutant protein does not

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cause a disease phenotype. In the hemoglobinopathies, for example, modifier genes play an important part in the appearance of clinical symptoms. Finally, the recognition that metabolic pathways are only rarely controlled by single rate-limiting steps greatly complicates the prediction of which symptoms a patient will develop based on the gene that is mutated. These possibilities all require further analysis in human patients as well as in model organisms before we can understand why the severity of monogenic diseases varies not only with different mutations in the same gene, but also among affected individuals within the same family.

21.6 The Genetic Background of Complex Disorders If there is a challenge to identify the genes involved in so-called monogenic diseases; clearly this challenge will be far greater for oligo- and polygenic disorders, which have multiple causes. Many of the diagnostic features of these complex diseases – called quantitative trait locus (QTL) disorders – are probably regulated by at least several genes. In monogenic diseases, mutations in a single gene are both necessary and sufficient to produce the clinical phenotype and to cause the disease (Fig. 21.1). The impact of the gene on genetic risk for the disease is the same in all families. In complex disorders with multiple causes, variations in a number of genes encoding different proteins result in a genetic predisposition to a clinical phenotype. Pedigrees reveal no Mendelian inheritance pattern, and gene mutations are often neither sufficient nor necessary to explain the disease phenotype. Environment and life-style are major contributors to the pathogenesis of complex diseases. In a given population, epidemiological studies expose the relative impact of individual genes on the disease phenotype. However, between families the impact of these same genes might be totally different. In one family, a rare gene C (Family 3) might have a large impact on genetic predisposition to a disease. However, because of its rarity in the general population, the overall population effect of this gene would be small. Some genes that predispose individuals to disease might have minuscule effects in some families.

21.7 Dissecting Interactions Between Genes and Environment Most common human diseases represent the culmination of lifelong interactions between our genome and the environment. Predicting the contribution of genes to complex disorders is still a challenge, and determining the interactions between genes and the environment during any disease process is a daunting task (Wu et al., 2008). Many human diseases, such as hypertension, coronary artery disease, and even some psychiatric disorders, represent quantitative traits that are caused by interactions among genes and between genes and the environment. The functional importance of identified DNA variations can be established by taking advantage of experimental opportunities in other species – for example, by introducing specific

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Fig 21.1 Trends of inheritance pattern and genetic risk in monogenic and complex disorders

DNA variations into animal strains that have a well-defined genetic background or that live in an environment that can be precisely controlled. The genes that contribute to complex disease are difficult to identify, because they typically exert small effects on disease risk; in addition, the magnitude of their effects is likely to be modified by other unrelated genes as well as environmental factors. Perhaps reflecting these difficulties, susceptibility loci for complex diseases identified in one study population often cannot be replicated in other populations. There is no unified story in genetic analysis of complex diseases.

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We will now consider seven diseases that offer contrasting examples of genetic determination. The selection is more or less arbitrary. We could equally well have chosen other diseases, but our examples illustrate some typical situations. We have not concentrated on presenting success stories. In some causes there is little progress to report.

21.7.1 Alzheimer Disease Alzheimer disease is the most common form of progressive dementia in the elderly. It is a neurodegenerative disorder characterized by the neuropathologic findings of intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques that accumulate in vulnerable brain regions (Sennvik et al., 2000). Within the higherfunctioning portions of their brains (the areas responsible for thought and memory), twisting tangles of threads made up of chains of tiny “tau” proteins are being assembled inside billions of nerve cells (neurons). Outside the neurons, other amyloid-beta (AB) proteins are fusing together into sticky clumps (plaque) akin to the substance that clogs heart arteries. Together, these tangles and plaques disrupt the normal functioning of the nerve cells, destroying the pathways along which packets of chemical “information” move. Memories cannot be stored or retrieved, and, eventually, the brain cannot control the body. The axonal defects studied in the mouse model were found to be similar to the axonal defects in the early stages of the Alzheimer disease in humans (Stokin et al., 2004). Bian et al. (2005) found no association of 6 A2M gene polymorphisms with Alzheimer disease in a study of 216 late-onset Alzheimer’s disease (AD) patients and 200 control subjects from the Han Chinese population. An estimated 26.6 million people worldwide had Alzheimer’s in 2006; this number may quadruple by 2050. The diagnosis of the disease is very difficult leading to its progress for years before getting fully diagnosed. Diagnosis is with the use of the diagnostic aids like medical imaging with computed tomography, magnetic resonance imaging, single photon emission computed tomography and positron emission tomography. The search for other disease loci gathered pace following the recognition of the genetic heterogeneity of AD. One of the early genetic investigations of Familial Alzheimer’s disease (FAD) that failed to show linkage to Chromosome 21 used a mixture of families, but the majority were of late onset (mean age: >60 years). Recently, several independent lines of evidence have provided support for the hypothesis that the disease locus on Chromosome 19 is the apolipoprotein E (ApoE) gene. ApoE is one of the many different proteins found to associate with A4 amyloid fibrils. Daw et al. (2000) estimated the number of additional quantitative trait loci (QTLs) and their contribution to the variance in age at onset of AD, as well as the contribution of apoE and sex. They also estimated that the apoE genotype can make a difference of as many as 17 years in age on the onset of the Alzheimer disease. The study in the Arab population in the Wadi Ara region couldnot explain the prevalence of the Alzheimer disease in relation with the data on the ApoE4 allele (Bowirrat et al., 2002).

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Olson et al. (2001) reported convincing evidence of a major role for the APP locus in late-onset AD. They used a covariate-based affected-sib-pair linkage method to analyze the chromosome 21 clinical and genetic data obtained on affected sibships by the Alzheimer Disease Genetics Initiative of the National Institute of Mental Health. In a comparison of 59 unrelated patients with AD and over 1000 controls, it was found that a combination of low head circumference and presence of the ApoE4 allele strongly predicted earlier onset of Alzheimer disease (Graves et al., 1990). The study of the two different populations (Dominican Republic and Puerto Rico) suggested that ApoE4 allele was associated with a nearly two fold increased risk of Alzheimer disease (Romas et al., 2002). In further use of a covariate-based linkage method to reanalyze genome scan data, it was determined that a region on chromosome 20p, showed the same linkage pattern to very-late-onset AD as APP. Two-locus analysis provided evidence of strong epistasis between 20p and the APP region, limited to the oldest age group and to those lacking E4 alleles at the ApoE locus (Olson et al., 2002). Among 563 AD patients and 118 controls, it was found that presence of the ApoE4 allele was strongly associated with reduced cerebrospinal fluid (CSF) levels of beta-amyloid-42 in both patients and controls (Prince et al., 2004). Elkins et al. (2004) concluded that the ACE I allele is associated with an increased risk of late-onset AD, but noted that the risk is very small compared to the effects of other alleles, especially ApoE4. Holmans et al. (2005) performed linkage analyses on 28 sib pairs with late-onset AD. Linkage was observed with chromosome 21 for age-at-onset effects. He also in the same year observed suggestive evidence of increased identical by descent in APOE4 homozygotes on chromosome. Although the APOE epsilon 4 allele is the only proven genetic risk factor for the late form of the disease, genetic epidemiological studies suggest that other loci are also involved (Hoenicka, 2006). The cerebral amyloid angiopathy(CAA) associated with the Alzheimer was also found in the Down syndrome and it was reasoned that due to the amyloid precursor protein gene (APP) locus located on the chromosome 21q21 might be affected by the gene dosage alterations in the subset of the demented individuals (Rovelet et al., 2006). Alzheimer’s disease has been identified as a protein misfolding disease, or proteopathy, due to the accumulation of abnormally folded A-beta and tau proteins in the brains of AD patients. Itoh et al. (2001) noted that the tau-199 test exceeds both sensitivity and specificity over 85% as a sole biomarker of AD; however, they also noted that many of the non-AD tauopathy and degenerative dementias also showed increased tau-199 levels. Sunderland et al. (2003) suggested that CSF beta-amyloid and tau are biologic markers of AD pathophysiology and that the measures may have potential clinical utility in the future diagnosis of AD. Scott et al. (2003) considered age of onset as a covariant in the analysis of data from 336 markers in 437 multiplex white AD families, they concluded that the linkage to the regions 2q34 and 15q22 were linked to early onset alzheimer disease and very late onset alzheimer disease respectively. In a study it was that the CSF markers are particularly useful to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias (Blennow et al., 2001). In a study it was found that CSF markers

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may be useful as diagnostic aids (Sjogren et al., 2003), especially to discriminate early or incipient AD from age-associated memory impairment, depression, and some secondary dementias (Blennow and Vanmechelen, 2003). The work on the CSF for the diagnosis of Alzheimer lead to the conclusion that changes in CSF biomarkers occur early in the course of AD in most patients (Herukka et al., 2005). Mace et al. (2005) found a significant association between a C-T SNP in exon 14 of the ABCA2 gene and Alzheimer disease in a large case-control study involving 440 AD patients. Li et al. (2005) found the SNP in theAPBB2 gene and reported using this marker the association of late-onset Alzheimer disease was most pronounced in subjects with disease onset before 75 years of age. Sillen et al. (2006) conducted a genome wide linkage study on 188 individuals with AD from 71 Swedish families, using 365 markers (average intermarker distance 8.97 cm). The results suggested that the disorder in these families was tightly linked to the ApoE4 region (19q13). Reiman et al. (2007) used a genome wide SNP survey to examine 1411 individuals with late-onset AD and controls, including 644 carriers of the ApoE4 allele and 767 noncarriers. There was a significant association between AD and 6 SNPs in the GAB2 gene that are part of a common haplotype block. An in vivo correlate of pathologic involvement, structural imaging measures are potential surrogate markers for disease progression in patients with established AD and in patients with prodromal AD, who will benefit most from disease-modifying therapies underway (Kantarci and Jack, 2003). It has been observed that simple MRI biomarkers, in addition to their diagnostic use, have a prognostic value with respect to mortality in a memory clinic population. A number of neuroimaging candidate markers are promising, such as hippocampus and entorhinal cortex volumes, basal forebrain nuclei, cortical thickness, deformation-based and voxel-based morphometry, structural and effective connectivity by using diffusion tensor imaging, tractography, and functional magnetic resonance imaging. CSF Abeta42, BACE1, total tau, and p-tau are substantially altered in MCI and clinical AD. Other interesting novel marker candidates derived from blood are being currently proposed (phase I). Biomarker discovery through proteomic approaches requires further research. Large-scale international controlled multicenter trials (such as the U.S., European, Australian, and Japanese Alzheimer’s Disease Neuroimaging Initiative and the German Dementia Network) are engaged in phase III development of the core feasible imaging and CSF biomarker candidates in AD. Biomarkers are in the process of implementation as primary outcome variables into regulatory guideline documents regarding study design and approval for compounds claiming disease modification (Hampel et al., 2008; Henneman et al., 2008). Finckh et al. (2000) investigated the proportion of early- onset dementia attributable to known genes. They screened for mutations in 4 genes, PSEN1, PSEN2, APP, and the prion protein gene PRNP, in patients with early-onset dementia before age 60 years. In 12 patients, they found 5 novel mutations and 5 previously reported mutations that were all considered to be disease - causing. In a study it has been reported that a deletion polymorphism at intron 16 of the ACE gene are also associated with AD (Wakutani et al., 2002). Rossi et al. (2004) reported a family in

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which at least 6 members spanning 3 generations had Alzheimer disease and strokes associated with a heterozygous mutation in the APP gene. Edwards-Lee et al. (2005) reported an African American family in which multiple members spanning 3 generations had early-onset Alzheimer disease. In this two siblings who were tested were found to be heterozygous for the mutation in the APP gene. Rovelet Lecrux (2006) estimated that in their whole cohort of 65 Autosomal dominant early onset Alzheimer’s disease (ADEOAD) families, the frequency of the APP locus duplication was roughly 8% (5 of 65), which corresponds to half of the contribution of APP missense mutations to ADEOAD. Alzheimer’s disease is a progressive complex disorder, where genetic predisposition interacts with the environmental factors. With the current knowledge of the pathogenesis of the Alzheimer’s disease the pharmaceutical industry is focussing on the development of the novel strategies with an etiopathogenic orientation (Gupta et al., 2008).

21.7.2 Cancer Cancer is a group of many related diseases that begin in cells, the body’s basic unit of life. Normally, cells grow and divide to produce more cells only when the body needs them. Sometimes, however, cells become abnormal and keep dividing to form more cells without control or order, creating a mass of excess tissue called a tumor. Tumors can be malignant (cancerous) or benign (not cancerous). Cancer can begin in any organ or tissue of the body. The primary, or original, tumor is usually named for the part of the body or the type of tissue in which the cancer begins. The disease can spread (metastasize) from the primary tumor and form metastatic tumors in other parts of the body. For example, breast cancer cells can metastasize to the lungs and cause the growth of a new tumor. When this happens, the tumor in the lung is called metastatic breast cancer because it is composed of breast cancer cells, not lung cancer cells (Anieta, 2009). Cancer is a diverse class of diseases which differ widely in their causes and biology. The common thread in all known cancers is the acquisition of abnormalities in the genetic material of the cancer cell and its progeny. Research into the pathogenesis of cancer can be divided into three broad areas of focus. The first area of research focuses on the agents and events which cause or facilitate genetic changes in cells destined to become cancer. Second, it is important to uncover the precise nature of the genetic damage, and the genes which are affected by it. The third focus is on the consequences of those genetic changes on the biology of the cell, both in generating the defining properties of a cancer cell, and in facilitating additional genetic events, leading to further progression of the cancer. Some cancers can be caused by infection with pathogens. Many cancers originate from a viral infection; this is especially true in animals such as birds, but also in humans, as viruses are responsible for 15% of human cancers worldwide. The main viruses associated with human cancers are human papillomavirus, hepatitis B and hepatitis C virus, Epstein-Barr virus, and human T-lymphotropic virus. Most forms of cancer are “sporadic”, and

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have no basis in heredity. There are, however, a number of recognized syndromes of cancer with a hereditary component, often a defective tumor suppressor allele. Famous examples are: • certain inherited mutations in the genes BRCA1 and BRCA2 associated with an elevated risk of breast cancer and ovarian cancer • tumors of various endocrine organs in multiple endocrine neoplasia (MEN types 1, 2a, 2b) • Li-Fraumeni syndrome (various tumors such as osteosarcoma, breast cancer, soft tissue sarcoma, brain tumors) due to mutations of p53 • Turcot syndrome (brain tumors and colonic polyposis) • Familial adenomatous polyposis, an inherited mutation of the APC gene that leads to early onset of colon carcinoma. • Hereditary nonpolyposis colorectal cancer (HNPCC, also known as Lynch syndrome) can include familial cases of colon cancer, uterine cancer, gastric cancer, and ovarian cancer, without a preponderance of colon polyps. • Retinoblastoma, when occurring in young children, is due to a hereditary mutation in the retinoblastoma gene. • Down syndrome patients, who have an extra chromosome 21, are known to develop malignancies such as leukemia and testicular cancer, though the reasons for this difference are not well understood. At present number of tumor markers have been identified and they are playing an important role in the diagnosis of the various cancers (Table 21.1). 1. Genetic Testing Genetic testing for high-risk individuals is already available for certain cancer-related genetic mutations. Carriers of genetic mutations that increase risk for cancer incidence can undergo enhanced surveillance, chemoprevention, or risk-reducing surgery. Early identification of inherited genetic risk for cancer, along with cancer-preventing interventions such as surgery or enhanced surveillance, can be lifesaving for high-risk individuals. 2. The Other Diagnostic Techniques 1. Imaging techniques- X-ray, Ultrasonography, computed tomography, magnetic resonance imaging. 2. Histopathology- studying of the tissue section of the affected tissue or cell. 3. Blood test- This is especially done for the diagnosis of the leukemia. 4. Biopsy- This involves the surgical removal of the affected organ or the tissue. 3. Molecular Marker Analysis Moul (1999) has shown that even though p53 and Bcl-2 are predictive biomarkers when sampling the radical prostatectomy specimen, they are not useful to predict postoperative recurrence when sampling the pretreatment needle biopsy. They also concluded that more research is needed to assess new biomarkers and, most importantly, to standardize the methodology for sampling and assaying biomarkers in heterogeneous and multifocal prostate cancer.

Description

Thyroglobulin

Prostate-specific antigen (PSA)

After surgery for colon cancer, texting can be useful in monitoring treatment and detecting recurrence.

This text cannot be recommended for cancer screening.

This text cannot be recommended for cancer screening

May be used to monitor response to treatment of medullary thyroid cancer. This is not recommended for routine cancer screening.

This test cannot be recommended for cancer screening.

Testing can be useful in diagnosing such cancers and in monitoring treatment.

Testing can be useful in diagonosing these cancers and in monitoring treatement

Comment about texting

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This text cannot be recommended for cancer screening. However, it is useful in assessing prognosis and monitoring treatment, particularly for people with testicular cancer, melanomas, and lymphomas. Levels are raised in men with noncancerous (benign) Testing can be useful in screening for cancer and in monitoring enlargement of the prostate and ofter are considerably higher its treatment in men with prostate cancer. What constitutes a meaningfully abnormal level is somewhat uncertain, but men with an elvated PSA level should be evaluated further by a doctor. Elevated blood levels may occur in patients with thyroid cancer This text cannot be recommended for routine screening but or benigh thyroid conditions. may be helpful for monitoring response to treatment of thyroid cancer.

Levels may be raised in blood of people with cancer of the colon. Blood levels may also be elevated in patients with other cancers or noncancerous conditions Beta-human chorionic This hormone is produced during pregnancy but also occurs in gonadortropin (β-HCG) women who have a cancer originating in the placenta and in men with various types of testicular cancer. Beta2 (β2)-microglobulin Levels may be raised in people with multiple mycloma or other cancers of blood cells. Calcitonin Produced by certain cells in the thyroid gland (C cells). Blood levels elevated in medullary thyroid cancer. Carbohydrate antigen 125 Levels may be increased in women with a variety of (CA-125) gynecological diseased, including ovarian cancer. Carbohydrate antigen 19-9 Levels may be increased raised in people with cancers of the (CA 19-9) digestive tract, particularly pancreatic cancer. Carbohydrate antigen 27.29 Levels may be increased in people with breast cancer. (CA27.29) Carcinoembryonic antigen Levels may be raised in the blood of people with cancer of the (CEA) colon. Blood levels may also be elevated in patients with other cancers or noncancerous conditions. Lactate dehydrogenase Levels can be raised for a variety of reasons.

Alpha-fetoprotein (AFP)

Tumor marker

Table 21.1 Selected tumor markers in diagnosis of disease 358 Diagnosis of Complex Diseases

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Bast et al. (2005) used two proteomic approaches: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Badgwell and Bast (2007) worked on the early detection of the ovarian cancer and measured multiple serum markers simultaneously. Peracaula et al. (2008) focused on the glycan changes of two serum glycoproteins, prostate specific antigen – currently used as a tumour marker of prostate cancer – and human pancreatic ribonuclease in pancreatic adenocarcinoma. The detection of glycan changes, associated with subsets of glycoforms in serum glycoproteins that are specific to the tumour situation, could be the basis for developing more specific biomarkers. Researchers at the University of Southern California (USC) and USC/Norris Comprehensive Cancer Center have identified genetic markers in cancer cells that predicted the benefit of a novel cancer drug prior to chemotherapy (Wilson et al., 2008). About 111 prostate cancer patients with bone metastasis at the diagnosis were enrolled. Thirteen genetic polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism or an automated sequencer with a genotyping software. There prognosis suggested the metastasis to be influenced by intrinsic genetic factors (Hansel, et al., 2009). The IGF-I (CA) repeat and CYP19 (TTTA) repeat polymorphisms may be novel markers in prostate cancer patients (Norihiko et al., 2007) with bone metastasis at the diagnosis (Tsuchiya et al., 2006). HPV testing has been suggested for primary screening, triage of equivocal Pap smears or low-grade lesions and follow-up after treatment for cervical intraepithelial neoplasia. Determination of HPV genotype, viral load, integration status and RNA expression could further improve the effectiveness of HPV-based screening and triage strategies (Boulet et al., 2008). Wang et al. (2009) studied on the systematic investigation of the differentially expressed genes during carcinogenesis in hepatocellular carcinoma (HCC) using cDNA microarray technology. This study describes a gene expression profiling of HCC, which provides an extensive list of potential molecular markers for early diagnosis and molecular targets for the development of drugs to treat patients with primary HCC. A significant correlation has been found between EGFR overexpression and PCNA labeling index in Grade III and Grade II astrocytomas and glioblastoma. These suggest that the tumor proliferation, at least in higher grades of astrocytomas is dependent in some measure on EGF and EGFR-related signaling pathways (Maiti et al., 2008). PMS2 protein levels have been shown to be a predictor of time-torecurrence after surgery. It is reported that the elevation of a mismatch repair protein negatively correlates with prognosis and has implications in patient diagnosis and molecular profiling (Norris et al., 2009). MRI has a role in well defined indications, but its role is still controversial even in some of these indications. MRI is not indicated in diagnosis in which image-guided percutaneous biopsy is the method of choice. Further studies must be performed (Van Goethem et al., 2009).

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In a study the effect of the TSP2 gene introduction into the Human malignant melanoma cell lines A375 was done and it was concluded that the TSP2 suppresses hematogenous metastasis through microenvironment-modification including PAI up-regulation and anti-vascularization in human malignant melanoma. Thrombospondin (TSP) 2 interacts with matrix metalloproteinases (MMPs) and matrix serine proteases such as plasminogen activator (PA) (Chijiwa et al., 2009). Cancer Researchers at the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch and the Charité – Universitäts Medizin Berlin (Germany) have identified a gene which enables them to predict for the first time with high probability if colon cancer is going to metastasize. They demonstrated that the gene MACC1 (Metastasis-Associated in Colon Cancer 1) not only promotes tumor growth but also the development of metastasis (Ulrike et al., 2009). When MACC1 gene activity is low, the life expectancy of patients with colon cancer is longer in comparison to patients with high MACC1 levels (Stein et al., 2009). There is no convincing evidence that changing dietary pattern following breast cancer diagnosis will improve prognosis for most women with early stage breast cancer. However, it would appear to be important for some subgroups. Further investigation of mechanisms for such selective action is needed (Pierce, 2009). The developments in the methods of the diagnosis and even more the in depth knowledge of the genetic level of the various types of the cancer has made it possible for the designing of the novel drugs, which aim at the precise cause of the disease.

21.7.3 Multiple Sclerosis Multiple sclerosis (abbreviated MS, also known as disseminated sclerosis or encephalomyelitis disseminata) is an autoimmune condition in which the immune system attacks the central nervous system, leading to demyelination. Disease onset usually occurs in young adults, and it is more common in women. MS affects the ability of nerve cells in the brain and spinal cord to communicate with each other. Nerve cells communicate by sending electrical signals called action potentials down long fibers called axons, which are wrapped in an insulating substance called myelin. In MS, the body’s own immune system attacks and damages the myelin. When myelin is lost, the axons can no longer effectively conduct signals. The name multiple sclerosis refers to scars (scleroses – better known as plaques or lesions) in the white matter of the brain and spinal cord, which is mainly composed of myelin. Although much is known about the mechanisms involved in the disease process, the cause remains unknown. Theories include genetics or infections. Different environmental risk factors have also been found. Almost any neurological symptom can appear with the disease, and often progresses to physical and cognitive disability. MS takes several forms, with new symptoms occurring either in discrete attacks (relapsing forms) or slowly accumulating over time (progressive forms). Between attacks, symptoms may go away completely, but permanent neurological problems often occur, especially as the disease advances. There is no known cure for MS. Treatments attempt to return

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function after an attack, prevent new attacks, and prevent disability. MS medications can have adverse effects or be poorly tolerated, and many patients pursue alternative treatments, despite the lack of supporting scientific study. The prognosis is difficult to predict; it depends on the subtype of the disease, the individual patient’s disease characteristics, the initial symptoms and the degree of disability the person experiences as time advances. Life expectancy of patients is nearly the same as that of the unaffected population. MS affects over 400,000 people in the United States and may affect 2.5 million people worldwide. Multiple sclerosis affects 2–3 times as many women as men. Further indications that more than one gene is involved in MS susceptibility comes from studies of families in which more than one member has MS. Several research teams found that people with MS inherit certain regions on individual genes more frequently than people without MS. Of particular interest is the human leukocyte antigen (HLA) or major histocompatibility complex region on chromosome 6. HLAs are genetically determined proteins that influence the immune system. The HLA patterns of MS patients tend to be different from those of people without the disease. Investigations in northern Europe and America have detected three HLAs that are more prevalent in people with MS than in the general population. Studies of American MS patients have shown that people with MS also tend to exhibit these HLAs in combination-that is, they have more than one of the three HLAs-more frequently than the rest of the population. Furthermore, there is evidence that different combinations of the HLAs may correspond to variations in disease severity and progression. Studies of families with multiple cases of MS and research comparing genetic regions of humans to those of mice with EAE suggest that another area related to MS susceptibility may be located on chromosome 5. Other regions on chromosomes 2, 3, 7, 11, 17, 19, and X have also been identified as possibly containing genes involved in the development of MS. These studies strengthen the theory that MS is the result of a number of factors rather than a single gene or other agent. Development of MS is likely to be influenced by the interactions of a number of genes, each of which (individually) has only a modest effect. Additional studies are needed to specifically pinpoint which genes are involved, determine their function, and learn how each gene’s interactions with other genes and with the environment make an individual susceptible to MS. In addition to leading to better ways to diagnose MS, such studies should yield clues to the underlying causes of MS and, eventually, to better treatments or a way to prevent the disease. Rosati (2001) did systematic study of multiple sclerosis in populations. Because the environmental and genetic determinants of geographic gradients are by no means mutually exclusive, the race versus place controversy is, to some extent, a useless and sterile debate. There is a small but significant north-to-south gradient of MS prevalence rates in Japan, suggesting that environmental factors varying with latitude also play a role in developing MS even in the low prevalence areas. Conventional MS in Japanese is, like MS in Caucasians, associated with HLADRB1∗ 1501 whereas opticospinal MS is associated with HLA-DPB1∗0501 (Kira,

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2003). Dharmasaroja (2003) reviewed and focused on the specificity of autoantibodies to the epitopes of myelin proteins and correlate this to the structures of proteins. Factors that influence the expression of myelin-protein epitopes such as the alpha-helical or beta-sheet structure of the protein, the tri-proline site, and the post-translational modifications as well as physicochemical properties of amino acid changed are included. Despite the number of issues that still need to be solved, the measurement of brain atrophy by MRI is sufficiently precise and accurate. It represents one of most promising in vivo measures of neuroaxonal degeneration in MS, and it should be used extensively in the future to assess and monitor pathological evolution and treatment efficacy in this disease (Stefano et al., 2007). With increased sophistication and improved analysis of these techniques of NMR and MRI scanning, understanding of the pathology underlying MS may increase, and objective quantification of the natural history of MS is possible (Rashid and Miller, 2008). MR imaging has a major impact on understanding the dynamic neuropathologic findings of multiple sclerosis (MS), early diagnosis of the disease, and clinical trial conduct. With new ways of monitoring the disease, new treatment targets should become practical, helping to translate advances in the understanding of immunology and regenerative medicine into novel therapies (Matthews, 2009). Bielekova et al. (2004) reviewed the general concepts of biomarkers and their potential use as surrogate endpoints and tailor these concepts to specific applications in multiple sclerosis research. Recent studies have pointed out new interesting candidates, driving new energies and hopes in the MS research community using the immunological markers (Rinaldi and Gallo, 2005). Reindl et al. (2006) concluded that antibodies serving as biomarkers will help to establish a differential therapeutic concept in MS, which will allow to treat individuals selectively according to their pathogenetic subtype and disease status. Berger and Reindl (2007) reviewed the current status and potential applicability of antibodies as biological markers for the diagnosis, classification, disease activity and prediction of clinical courses in MS. Lutterotti et al. (2007) worked on the identification of biomarkers for MS in cerebrospinal fluid and/or blood. They focussed on antibodies to myelin and nonmyelin antigens, cells and soluble molecules of the immune system and the brain as biomarkers for (1) the diagnosis and prediction of clinical courses, (2) disease activity and (3) treatment response in MS. Wu et al. (2008) systematically evaluated a series of stilbene derivatives as myelin imaging agents. Spectrophotometry-based and radioligand-based binding assays showed that these stilbene derivatives exhibited relatively high myelin-binding affinities. These studies suggested that these stilbene derivatives can be used as myelin-imaging probes to monitor myelin pathology in vivo. Satoh (2008) identified the principal molecular network involved in development of MS and induction of acute relapse. Thus, DNA microarray technology is highly valuable to identify molecular mechanism-based biomarkers (Bibiana and Roland, 2004) for classification of MS subgroups and prediction of MS relapse. Autoantibody profiles against epitopes derived from MS brain tissue could serve as diagnostic markers or form the basis for the identification of a subgroup of MS patients (Somers et al., 2008). Anti-MBP autoantibody-mediated, epitope-specific

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binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression (Belogurov et al., 2008).

21.7.4 Diabetes Diabetes and its associated complications have affected about 200 million people worldwide representing 6% of the population. Diabetes is a disorder of metabolism. Diabetes mellitus (DM) is one of the most common chronic diseases in children and adolescents, and type II DM accounts for more than 95% of cases. Nevertheless, over the last years it has become apparent that not all cases of DM presenting in children have an autoimmune basis. In addition to type 2 DM, which continues to be an infrequent diagnosis among pediatric patients in most countries worldwide, several forms of monogenic DM may present during childhood and are responsible for the disease in 1–3% of patients. Molecular diagnosis, increasingly available, improves both clinical management and quality of life, and is also important for genetic counselling (Rubio-Cabezas and Argente, 2008). Genetic alterations in expression profiles of pancreatic cells result in phenotypic changes. Therefore, mapping the chromosomal location of the genes that encode differentially expressed transcripts found by microarrays, may lead to the identification of underlying genetic differences accounting for diabetes. The generation of transcriptome maps for tissues from diabetic and non -diabetic individuals could provide tools to identify candidate genes that are overexpressed or silenced in this disease. Cell-based therapies offer the possibilities of a permanent cure for diabetes. Recently, success in the transplantation of pancreatic islets in the pancreas of type 1 diabetics has afforded the opportunity for a potential cure. Now, the genes underlying certain forms of diabetes are being discovered, and are having a direct impact on treatment. Type 2 diabetes is a classic “polygenic” disease, caused by the interaction of multiple genes and the environment. But this is not always the case: in 1–2% of cases, a mutation in a single gene can cause diabetes. The commonest type of single gene diabetes was originally classified clinically as “maturity onset diabetes of the young” (MODY). This is a form of young-onset diabetes (typically diagnosed under 25) that is not insulin-dependent and is inherited. Since 1992, mutations in six genes have been found to cause MODY, most cases involving the enzyme glucokinase or one of three gene-regulatory proteins: hepatic nuclear factor-1a (HNF-1a), HNF-4a and HNF-1b. Mutations in these genes affect insulin production by the beta cells, but the outcomes are markedly different. People with glucokinase mutations are born with mildly raised blood glucose levels, and their condition deteriorates little throughout their life. With a careful diet, there is usually no need for insulin injections or drug treatment. People with mutations in one of the MODY transcription factors are usually born with normal blood glucose but usually develop diabetes between the ages of ten and 25. In fact, they are often misdiagnosed as having type 1diabetes, as they are in the right age range, are slim and can have high blood glucose levels.

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Busch and Hegele (2001) studied many common variants in functional and positional candidate genes, including ADRB3, PPARG, ENPP1, and CAPN10, and also evaluated for their possible role as determinants of type 2 diabetes. Ksiazek et al. (2003) suggested that the ecNOS gene polymorphism can serve as a useful genetic marker of increased susceptibility to type 2 diabetes and its renal complications. Wasserfall and Atkinson (2006), worked on the four autoantibodies: islet cell cytoplasmic (ICA), insulin (IAA), glutamic acid decarboxylase (GADA), and IA2/ICA512 autoantigen (IA2A), for the detection of diabetes 1 and found that these markers combined with other metabolic and genetic markers, are extremely effective for predicting eventual development of type 1 diabetes in otherwise healthy individuals. In a study it was demonstrated that adiponectin was an independent predictor for gestational diabetes mellitus (GDM). As for GDM screening, adiponectin was not as strong a marker as glucose challenge test (GCT). However, with advantage of being less cumbersome, adiponectin could be used to rule out pregnant women at low risk of GDM (Weerakiet et al., 2006). Eyzaguirre and Mericq (2009) worked on the various currently known biochemical markers to evaluate insulin sensitivity and asses their utility as markers. McCarthy and Hattersley (2001) surveyed present knowledge about the genetic basis of Type 2 diabetes and discussed the current and future role of genetic diagnostics, with an emphasis on lessons learned from study of monogenic forms of the disease. Hansen (2009) studied and supported the candidate gene approach as a feasible method for directly either identifying or excluding any gene as a diabetessusceptibility gene (“diabetogene”). Broedl et al. (2006) studied the genetic aspect of the diagnosis of diabetes and concluded that genetic tests are crucial for the correct classification of the type of diabetes, genetic counseling, and initiation of the appropriate therapy regimen. Recent study demonstrated that M55V variant in SUMO4 at IDDM5 was associated with susceptibility to type 1 diabetes in the Asian population (Noso et al., 2006). Molecular analysis of chromosome 6 anomalies, the KCNJ11 and ABCC8 genes encoding Kir6.2 and SUR1 provide a tool to identify transient from permanent neonatal diabetes mellitus in the neonatal period (Flechtner et al., 2007). ABCC8 mutations cause PNDM, TNDM or permanent diabetes diagnosed outside the neonatal period. There is some evidence that the location of the mutation is correlated with the clinical phenotype (Patch et al., 2007). Among the world’s most rapidly spreading complex diseases is the diabetes. The exact genetic cause of the diabetes is not yet established and the latest research has lead to the multiple gene linkage of the disease and the better understanding of the disease. The biotechnology holds the future for the possible cure for the disease as stem cell therapy and the recombinant insulin with even more efficiency are in line to be approved by the governing bodies.

21.7.5 Hemophilia Hemophilia is a bleeding disorder that slows the blood clotting process. People with this condition may experience prolonged bleeding or oozing following an injury, surgery, or having a tooth pulled or spontaneous bleeding in joints and muscles

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in severe cases of hemophilia. Serious complications can result from bleeding into the brain, abdomen or other internal organs. The major types of this condition are hemophilia A (also known as classic hemophilia) and hemophilia B (also known as Christmas disease). Although the two types have very similar signs and symptoms, they are caused by mutations in different genes. Both are X linked disorders and hence, manifest predominantly in males. Female carriers of the mutation are usually asymptomatic. The two major forms of hemophilia occur much more commonly in males than in females. Hemophilia A is characterized by deficiency in factor VIII clotting activity. Hemophilia B is inherited in an X-linked recessive pattern. Hemophilia B is characterized by deficiency in factor IX clotting activity. The age of diagnosis and frequency of bleeding episodes are related to the level of factor VIII and IX clotting activity. The diagnosis of hemophilia B is established in individuals with low factor VIII and IX clotting activity. Molecular genetic testing of F8 and F9, the gene encoding factor VIII and IX, identifies disease-causing mutations in more than 99% of individuals with hemophilia A and B. Detection of mutation is useful for the carrier detection and prenatal diagnosis (Ljunj 1996). As the number of the mutations is very high, mutation detection is difficult for the clinical purposes. Hence, for carrier detection of hemophilia in females at risk, Restriction fragment length polymorphism (RFLP) linkage analysis is most widely used. Linked markers are also used for the prenatal diagnosis. The two types of polymorphism, namely; single nucleotide polymorphisms (SNPs) and length polymorphisms, also known as variable number tandem repeat sequences (VNTRs) or microsatellites are commonly used for linkage analysis. Polymorphisms have a scientific interest of their own and are useful in areas as diverse as; gene mapping, forensic science and the study of human evolution. They have clinical relevance in the context of hereditary disorders in that they can be used to track a defective (or normal) gene through an affected family. Such linkage studies have permitted carrier status investigation and prenatal diagnosis in hemophilia A and hemophilia B. DNA analysis using intragenic or closely linked restriction fragment length polymorphisms (RFLPs) is rapidly gaining acceptance as a reliable and Practical approach to carrier detection in both hemophilia B and hemophilia A. It is easier and convenient than mutation detection by gene sequencing (Berber et al., 2006). High throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon-intron boundaries of the factor VIII gene was done in 89 hemophilia A patients and it was found that the correlation of phenotype with genotype as observed in this study was not absolute (Citron et al., 2002). Direct carrier testing was done in 54 at-risk female relatives of haemophilic patients by initially analysing 2·46 kb of the factor IX gene in 1 haemophiliac per family by genomic amplification with transcript sequencing. A presumptive mutation was found in all 14 haemophiliacs examined (Bottema et al., 1989). The technique has clinical application (Sorenson et al., 2003). Identification of a causative mutation leads to more precise carrier detection than does conventional polymorphism-based linkage analysis. This can effectively be used to establish genotype/ phenotype relationships (Mahajan et al., 2004). Jayandharan et al. (2005), analysed DNA from 109 unrelated Indian patients with HA for their

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FVIII gene defects, and were screened for point mutations by a multiplex PCR and conformation sensitive gel electrophoresis strategy. Pandey and Mittal (2001) demonstrated that the combine use of the RFLP markers and short tandem repeats can be used to identify carriers and provide prenatal diagnosis in hemophilia A upto 95% accuracy and help prevent the extent in the hemophilia A affected family. Using the polymorphic markers like intron 18 Bcl I, intron 19 Hind III, intron 22 xbaI and DXS52/St14 of the factor VIII gene and intron 1 DdeI, intron 4 TaqI, 3 HhaI and the residue 148 codon Mnl I of the factor nine gene, diagnosis of hemophilia can be made (Shetty et al., 2001). For firsttrimester diagnosis in the chorionic villus samples, we have used both direct and indirect methods, that is, intron 22 and 1 inversions in the factor VIII gene, a multiplex PCR for the detection of gross deletions in the factor IX gene and RFLP analysis using a battery of markers within and outside the factor VIII/IX gene. This method yielded positive result and hold a lot of promise for the prenatal diagnosis of haemophilia (Shetty et al., 2006). De Carvalho et al. (2007) used three microsattelite repeats and one HindIII RFLP markers for the carrier detection of the hemophilia in the brazilian population. Molecular analysis of 76 unrelated Iranian haemophilia B patients was performed by PCR, single strand conformational polymorphism (SSCP) on important functional regions of the F9 gene followed by sequencing on samples with different migration pattern. Using this approach we found mutation in 52 out of 76 patients (Karimipoor et al., 2008). Saha et al. (2007) examined variations of single nucleotide polymorphism (SNP) in F8 in the Indian population and established the utility of a combination of SNP and microsatellite markers for the successful identification of carriers in the affected families. Polymerase chain reaction (PCR) and restriction enzyme analysis were used to study the polymorphism in bcl1, and long-distance PCR for detection of VNTR (ST14) alleles. Carrier detection and prenatal diagnosis is possible in haemophilia A families using both DNA markers. They suggest screening haemophilic families first for bcl1 polymorphism followed by analysis of St14 locus (Hussein et al., 2008). Study was done on the 33 unrelated hemophilia B patients with PCR amplification of the exon sequence and multiplex ligation-dependent probe amplification (MLPA) was performed for the exon dosage test. It was concluded that MLPA can be a feasible platform at clinical laboratories to detect large deletion mutations in the suspected cases (Kwon et al., 2008). Kim et al. (2005) demonstrated the utility of two intragenic markers in intron 1 and 24 and the other markers intron 13 and 22, using fluorescent PCR in factor 8 gene for carrier detection and prenatal diagnosis of hemophilic families. Diagnostic tests were validated using previously studied samples. IS-PCR evaluated carrier mosaicisms and performed robustly over wide ranges of DNA qualities and procedural conditions. IS-PCR improved the molecular diagnosis of Hemophilia A. This genotyping strategy may potentially be adapted to virtually all known rearrangements in the human genome (Rossetti et al., 2008). High-resolution melting analysis is an appealing technology for F8 gene screening. It is rapid and quickly identifies mutations in the majority of HA patients; samples in which no mutation is detected require further testing by DNA

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sequencing. The LC480 and Light Scanner platforms performed similarly (Laurie et al., 2007). Haemophilia is a genetic disorder of the blood. The recent advancement in the field of diagnosis has lead to the early diagnosis of the disease at the foetal level and helps the parents with the family history of the disease to decide on their future with the help of the genetic counselling along with the diagnosis. The advancement (Rossetti et al., 2008) in the genetic level of the disease has helped in the development of the more targeted form of gene therapy.

21.7.6 Obesity Obesity is a condition of increased adipose tissue mass. Obesity can also be defined as an increase in body weight beyond the limits of physical requirement, as the result of an excessive accumulation of fat. Accumulation of fat, or triacylglycerol, is essentially the only way that body weight can become excessive, as other energy storage (e.g. carbohydrate glycogen or protein in liver and muscle) does not have the potential of adipose tissue to exceed the limits of requirement. Obesity is associated with a significant increase in morbidity and mortality and is a major public health problem. For reasons that are not fully known, obesity is associated with an increased risk of hypertension, heart disease, diabetes and cancer. Even modest weight loss ameliorates these associated conditions. In addition to the prospect of diminished health, obese people are often stigmatized both socially and in the workplace. Although the premium on leanness has become especially prominent in late twentieth-century Western societies (at least among the affluent), this view is very dependent on the cultural context. In many cultures obesity is considered to be a sign of affluence and prestige, particularly among those cultures where food is less available. In modern times, however, intense pressure to be thin is felt by most individuals, lean and obese. Despite this, obesity affects a significant and increasing number of individuals. The heritability of obesity is roughly equivalent to that of height and exceeds that of many disorders that are generally considered to have a genetic basis. The identity of several of these genes is now known and in these instances the evidence that obesity is not simply a personal failing is overwhelming (James and Trayhum, 1976). In search for the genetic etiology of the obesity-related metabolic adversities, phenotyping and genotyping of individuals from 507 Caucasian families were conducted. An obese proband was identified by quantitative trait loci (QTLs) on chromosomes 3 (3q27) and 17 (17p12), which were strongly linked to variation in total body adiposity, abdominal fat partitioning, and insulin resistance. The strong evidence for a genetic component to human obesity has been unequivocally established over the past years with the identification of the genetic defects responsible for different monogenic forms, being involved in 4% of cases of human obesity (Clement et al. 2002). However, the role of genetic factors in common obesity is complex, being determined by the interaction of several genes (polygenic), each of which may have relatively small effects (i.e., they are susceptibility genes) and

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which may work in combination with each other as well as with environmental factors (e.g., nutrient intake and physical activity). The examination of candidate genes for involvement in the susceptibility to common obesity has not yet yielded convincing results (Chagnon et al. 2003). Another approach used for identifying genes underlying common polygenic obesity utilizes genome-wide scans in order to detect chromosomal regions showing linkage with obesity in large collections of nuclear families. The power of this approach was proven in other complex traits (Horikawa et al. 2000; Gretarsdottir et al. 2003). In another study, a genome-wide scan performed in 158 multiplex French obese Caucasian families (514 individuals) having at least one subject with a body mass index (BMI) of greater than 40 kg/m2 and at least one further sibling with a BMI of greater than 27 kg/m2 reported significant evidence for linkage of obesity to a Chromosome 10p locus (Hager et al. 1998), with a maximal logarithm of odds (LOD) score (MLS) near the D10S197 marker was done. Replication studies in both European–American and African–American cohorts confirmed the maximum nonparametric linkage peak at D10S197 for the combined sample set (Price et al. 2001) as well as for a German young obese sib cohort (Saar et al. 2003). All together, these independent linkage studies strengthen the hypothesis of a susceptibility gene for obesity in the Chromosome 10p11–12 locus. Marker D10S197 is located in intron 7 of the GAD2 gene encoding the glutamic acid decarboxylase enzyme (GAD65). GAD65 catalyzes the formation of γ-aminobutyric acid (GABA) from L-glutamic acid and is expressed in both pancreatic islets and brain (Erdo and Wolff, 1990). GABA is colocalized in neuropeptide Y (NPY) neurons and is involved in the leptin pathway through the arcuate nucleus in the hypothalamus (Ovesjo et al. 2001). GABA interacts with NPY in the paraventricular nucleus to stimulate food intake (Pu et al. 1999). The bilateral injection of GAD2 antisense oligodeoxynucleotide into rat ventromedial hypothalamus decreased the content of GABA by 50% 24 h after the injection, decreasing food intake, while also enhancing locomotor activity (Bannai et al. 1998). The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11– 12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of γ-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake (Boutin et al., 2003). It can be concluded that genetic and functional arguments favor the GAD2 gene as a positional candidate gene for obesity on the Chromosome 10p locus. QTL on human chromosome 7q35–q36 that affects both plasma TG and LDLC variation in Caucasian families of predominantly northern European ancestry have also been localised. Several suggestive QTLs were also localized, including a region on chromosome 12p12.3, which affects plasma HDL-C variation. The large cohort of families studied, the selection of families including both obese and neverobese individuals, and the magnitude of the LOD scores identified emphasize the important contributions of these loci to the genetic architecture associated with the obesity-related lipid disorder.

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The additive genetic heritability estimates for the lipid-lipoprotein phenotypes analyzed in our cohort range from 0.30 to 0.47 and strongly suggest the existence of genetic factors, which influence expression of this adverse lipid profile. Similar degrees of heritability influencing plasma levels of the same lipid components have previously been reported in twin, adoption, and family studies in nonobese normal subjects and individuals with established coronary artery disease. Our confidence in the stability of the QTL on chromosome 7 (7q35–q36) is also heightened by its replication in other studies. In a cohort of Mexican-Americans, Duggirala et al. identified a locus harboring susceptibility genes for hypertriglyceridemia in this same region, while analysis of results from Caucasian families in the Framingham Heart Study provided evidence for a gene near this locus that affects the ratio of plasma TG and HDL-C. Moreover, a susceptibility locus for hereditary pancreatitis, a disorder frequently resulting in hypertriglyceridemia, has been mapped to this location. Li and Loos (2008) studied the genetic progress of obesity and concluded that Genome-wide association promises to enhance greatly our understanding of the genetic basis of common obesity, although candidate gene studies will remain a valuable approach because they allow more detailed analyses of biologically relevant candidates. A key factor contributing to continued success lies in large-scale data integration through international collaboration, which will provide the sample sizes required to identify genetic association with conclusive evidence. Pantsulaia et al. (2009) studied and suggested that genetic factors play a significant role in regulation of variation of the examined traits. The variation of fat distribution measures (OB) traits is almost fully due to genes influencing variation of adipokines (AC), whereas the correlation between blood pressure (BP) and AC is only marginally significant and caused only by shared environment. Studies in humans and rodents have suggested a central role for FTO through regulation of food intake, whereas others have proposed a peripheral role through an effect on lipolytic activity in adipose tissue (Loos and Bouchard, 2008). Recent advancement in scanning the loci, mapping to the fat mass and obesity associated gene (FTO), influences diabetes risk through a primary effect on fat mass, making this the first common variant known to influence weight and individual risk of obesity (Lindgren and McCarthy, 2008). Percy et al. (2008) suggested that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related Chronic Kidney disease (CKD). Gaillard et al. (2008) gave an overview of postoral regulation of food intake by lipids and then highlights recent data suggesting the existence of a “fatty taste” which might contribute to lipid overeating and hence to the risk of obesity. Armutcu et al. (2008) suggested that oxidative stress parameters and components of metabolic syndrome are closely related; therefore, significant alterations may occur in the antioxidant and inflammatory status. However, further studies are required to evaluate the possible molecular mechanisms of heat shock protein 70 levels in metabolic syndrome. Phipps et al. (2008) suggested that body size may also be related to the risk of postmenopausal triple-negative

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breast cancer among nonusers of hormone therapy. Given the expanding obesity epidemic, the widespread cessation of hormone therapy use, and the poor prognosis of triple-negative tumors, this novel finding merits confirmation. Scheja et al. (2008) identified high fat (HFD)-fed mice as a suitable model to study A-SAA as a biomarker and a novel possible mediator of insulin resistance. A study suggested that Peripheral blood mononuclear cells (PBMC) are a suitable RNA source and model system to perform nutrigenomics studies related to obesity and development of personalized dietary treatments. IL8 gene expression warrant further research as a putative novel biomarker of changes in body fat percentage in response to an low-Calorie diet (LCD) (Crujeiras et al., 2008). Obesity is a multiple symptomatic disease, which is characterized mainly by the overweight of the patient. Other symptoms arise due to malfunction in the metabolic processes. Its genetic study can help identify the genes responsible and their possible treatment, by treating the error in the metabolic pathway.

21.7.7 Schizophrenia Schizophrenia is a chronic, severe, and disabling brain disease. It is found all over the world. The severity of the symptoms and the long-lasting, chronic pattern of schizophrenia often results in disability, and many patients need ongoing assistance to manage the most basic functions of independent living. People with schizophrenia may have perceptions of reality that are strikingly different from the reality seen and shared by others around them. Their behavior may seem odd, unusual or even bizarre at times. They sometimes hear voices, talk to themselves, or respond to imaginary fears. The recent study has evaluated various risks factors causing schizophrenia. At times, normal individuals may feel, think, or act in ways that resemble schizophrenia. Normal people may sometimes be unable to “think straight.” They may become extremely anxious, for example, when speaking in front of groups and may feel confused, be unable to pull their thoughts together, and forget what they had intended to say. This is not schizophrenia. At the same time, people with schizophrenia do not always act abnormally. Indeed, some people with the illness can appear completely normal and be perfectly responsible, even while they experience hallucinations or delusions. An individual’s behavior may change over time, becoming bizarre if medication is stopped and returning closer to normal when receiving appropriate treatment. There is a common misconception that schizophrenia is the same as a “split personality”. Another related misconception is that schizophrenia results in several different personalities, and the individual switches between these different persons. These perceptions are not correct. Although schizophrenia affects men and women with equal frequency, the disorder often appears earlier in men, usually in the late teens or early twenties, than in women, who are generally affected in the twenties to early thirties. People with schizophrenia often suffer terrifying symptoms such as hearing internal voices not heard by others, or believing that other people are reading their minds, controlling

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their thoughts, or plotting to harm them. These symptoms may leave them fearful and withdrawn. Their speech and behavior can be so disorganized that they may be incomprehensible or frightening to others. Available treatments can relieve many symptoms, but most people with schizophrenia continue to suffer some symptoms throughout their lives; it has been estimated that no more than one in five individuals recovers completely. This is a time of hope for people with schizophrenia and their families. Research is gradually leading to new and safer medications and unraveling the complex causes of the disease. Scientists are using many approaches from the study of molecular genetics to the study of populations to learn about schizophrenia. Methods of imaging the brain’s structure and function hold the promise of new insights into the disorder. In follow-up from evidence obtained in linkage studies, systematic linkage disequilibrium mapping within chromosomal region 13q33 has led to the identification of a schizophrenia susceptibility locus which harbors the genes G72 and G30. These association findings have been replicated in several independent schizophrenia samples. Association has also been found between genetic variants at the G72/G30 locus and bipolar affective disorder (BPAD), with replication in independent studies. Results from studies of more detailed psychiatric phenotypes show that association exists with symptom clusters that are common to several disorders as well as with specific psychiatric diagnoses. These findings may indicate that the association lies not with the diagnostic categories per se but with more specific aspects of the phenotype, such as affective symptoms and cognitive effects, which cross traditional psychiatric diagnostic boundaries. At the molecular level, the picture remains far from clear. No putative functional variants have been identified in the coding regions of G72 or G30, and it is therefore likely that disease susceptibility is caused by as yet unidentified variants which alter gene expression or splicing. A further complication is the fact that inconsistencies are evident in the risk alleles and haplotypes observed to be associated across different samples and studies, which may suggest the presence of multiple susceptibility variants at this locus. Functional analyses indicate that the G72 gene product plays a role in the activation of N-methyl-D-aspartate receptors, a molecular pathway implicated in both schizophrenia and BPAD, making it the most plausible candidate gene at this locus. Three differentially expressed proteins in schizophrenia have been identified viz. manganese superoxide dismutase (MnSOD) was under-expressed; and collapsing response mediator protein 2 (CRMP-2) and t-complex protein 1 (TCP-1) were over-expressed. MnSOD catalyzes the dismutation of superoxide anion (O2 – ) into water (H2 O) and hydrogen peroxide (H2 O2 ). In nervous tissue, it may protect the survival of cell membranes. CRMP-2 regulated axonal growth and polarity. Its over-expression may explain the vast neural interconnections in the schizophrenic brain. Oxidative stress is particularly implicated in neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases. Gene expression studies with schizophrenia have consistently identified oxidation related transcripts. Theories have postulated that when the production of harmful oxidants exceeds the rate of anti-oxidant compounds, macromolecules such as DNA and proteins become the targets of oxidative

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attack, which signals subsequent death. Moreover, Japanese scientists were the first to identify proteins targets for nitration in the brains of rats, which included TCP-1. The original research methodology did not allow for post-translational modification testing; the state of TCP-1 in schizophrenic hippocampal tissue therefore remains unknown. Chromosomal location is another feature of the study that sheds light on schizophrenia. Three of the four characterized proteins were mapped to chromosomal arm 6q. Their vicinity reveals a region important in schizophrenia and confirms the susceptibility of loci found by the linkage study. Despite the informative studies using 2D-GE with schizophrenia, this modality proved very limited. Three important issues were raised that have instigated investigations into other resolution measures. First, 2D-GE analysis has limited reproducibility. Second, weakly soluble proteins cannot be easily resolved. Third, only a tiny portion of the proteome can be effectively stained. It is especially difficult for low-level expressed proteins and those masked by greater expression within a similar molecular weight or isoelectric point, or both. Heritability estimates for schizophrenia range from 70 to 80%. However, efforts to map susceptibility genes using traditional linkage studies have been inconsistent, probably because of complex patterns of inheritance and the relatively small risk conferred by individual genes. An alternative to linkage analyses is to test candidate genes for linkage and association using the transmission disequilibrium Test (TDT). Identification of such candidates has come more rapidly with the use of modern molecular methods, such as gene microarrays. The expression of RGS4, but not other RGS family members, is decreased across the cerebral cortex in subjects with schizophrenia, but not among subjects with major depressive disorder. RGS proteins function primarily as GTPase-activating proteins (GAPs) for heterotrimeric G-protein α(Gα ) subunits, accelerating the hydrolysis of Gα -bound GTP. Thus, they shorten the duration of intracellular signaling of many G-protein-coupled receptors (GPCRs) belonging to dopamine, GABA, glutamate and other neurotransmitter systems. RGS4 is an interesting candidate gene for additional reasons. It is highly expressed in brain regions implicated in the pathophysiology of schizophrenia, modulates function of multiple G-proteincoupled neurotransmitter receptors and exhibits robust transcriptional changes to stress. Furthermore, RGS4 maps to chromosome 1q21–q22, a region implicated in prior schizophrenia linkage studies. Emilien et al. (1999) studied the relationship between dopamine receptors and schizophrenia and lent some useful insights as to the potential advantages of the newer antipsychotics which appear to have a sparing or beneficial effect on various components of cognitive function. Golimbet (2008) studied the association between gene polymorphisms and cognitive dysfunctions in schizophrenic patients. The main emphasis was to the association between gene polymorphisms and cognitive dysfunctions in schizophrenic patients. The association studies on the genes for dopamine and serotonin receptors, brain-derived neurotrophic factor, dysbindin, DISC1, D-amino acid oxidase and D-amino acid oxidase activator are reviewed as well.

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Post-mortem studies have revealed a lower density of glutamatergic receptors in patients with schizophrenia. Other studies of cerebrospinal fluid reported lower levels of glutamate in patients with schizophrenia in healthy comparison subjects. The most compelling evidence is provided by the psychomimetic effects of the NMDA antagonists’ phencyclidine and ketamine. Pharmacological modulation of the effects of NMDA receptor antagonists, such as ketamine, may lead to development of novel therapeutic agents for psychiatric illnesses such as schizophrenia (Mechri et al., 2001). Bota et al. (2008) reviewed the development of the prodromal concept and its implications for early identification. Morrens et al. (2008), gave considerable evidence to support the existence of a cluster of psychomotor symptoms, in addition to positive, negative and cognitive symptoms. The highly sensitized computerized neuropsychological testing was used for monitoring of the cognitive effects of antipsychotics. Computerized assessment assists in the identification of state- and trait-related cognitive impairments (Kertzman et al., 2008). Nuechterlein et al. (2004) studied some schizophrenia patients and concluded that seven separable cognitive factors were replicable across studies and represent fundamental dimensions of cognitive deficit in schizophrenia: Speed of Processing, Attention/Vigilance, Working Memory, Verbal Learning and Memory, Visual Learning and Memory, Reasoning and Problem Solving, and Verbal Comprehension. In 2004 a meeting was held, based on discussions at this meeting, five criteria were considered essential for test selection of schizophrenia: good test-retest reliability, high utility as a repeated measure, relationship to functional outcome, potential response to pharmacologic agents, and practicality/tolerability (Green et al., 2004). Stip (2006) reviewed and concluded that from now on cognitive deficit should be recognized as a major element in social and professional integration of schizophrenia patients, and should become a standardized assessment approach in clinical practice. Schwarz and Bahn (2008) reviewed molecular alterations of proteins and metabolites that have been identified in schizophrenia CSF and discuss their potential applicability as diagnostic markers (Sheja et al., 2008). Chopra et al. (2008) studied various patents that describe the use of nanoparticles to deliver various neurotherapeutics and neurodiagnostics to brain. Advances in DNA technology have provided important new tools in the effort to discover the genetic basis of schizophrenia. One of the most valuable techniques is linkage analysis, which is based on the fact that, when chromosomes cross over and exchange segments of DNA (i.e. recombine) during meiosis, genetic loci that are close to each other are more likely to be co-inherited than are loci that are more distant. This is important to the identification of genes that cause disease because, if an unknown disease gene is “linked” (i.e. co-segregates) with a DNA marker having a known chromosomal location, the marker can be used to find the gene. This marker must be polymorphic (since variants must be specific to a disease). Linkage analysis became a powerful tool when molecular geneticists developed methods to identify many DNA markers throughout the genome.

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Statistical methods of linkage analysis involve computing the probability that the co-segregation of genetic markers and disease within pedigrees exceeds what would be expected by chance. Many linkage studies have been performed under SML conditions, in which the odds for linkage are analysed for a specified degree of linkage. If the odds against a random finding (expressed as the logarithm of odds ratio, or LOD score) exceed 1000 to 1, a LOD of 3 is assigned, and evidence of linkage between a gene and a trait is provided. On the other hand, a LOD score of – 2 is considered to be a cut-off point used to exclude the possibility of linkage. These statistical decision rules have proven to be reliable for single gene diseases; however, for complex disorders such as schizophrenia, other factors such as the presumed mode of transmission, the definition of the phenotype, the degree of penetrance, the sample size and the number of affected family members, must also be considered in determining linkage. Unfortunately, some of those factors, such as the mode of transmission and the degree of penetrance, are not yet known for schizophrenia. To compensate, linkage analyses can be performed repeatedly, with different values set for each parameter. However, a disadvantage of this approach is that positive results must be viewed conservatively, because the risk of false positive findings increases with the number of tests performed. In general, linkage analysis is most useful in uncovering variance in important genes that follow Mendelian patterns of inheritance; it is less helpful in identifying genes that exert a small or moderate effect upon complex psychiatric disorders. This problem is exacerbated when definitions of phenotypes are not accurate. Despite these reservations, evidence has been aggregated that link schizophrenia to several chromosomal sites (Chromosome 1, 5, 6, 8, 10, 11, 13, 15, 18, and 22). Schizophrenia is a complex psychiatric disorder, who’s diagnosis for long has been a challenge for the doctors and the scientists. The modern diagnostic methods have opened the new realm for the genetic study of the disease and the development of the psychotropic drugs, which can be even used in treating other psychological diseases.

21.8 Conclusion We are rapidly advancing upon the postgenomic era in which genetic information will have to be examined in multiple health care situations throughout the lives of individuals. Currently, newborn babies can be screened for treatable genetic diseases such as phenylketonuria. Perhaps in the not-so-distant future, children at high risk for coronary artery disease will be identified and treated to prevent changes in their vascular walls during adulthood. Parents will have the option to be told their carrier status for many recessive diseases before they decide to start a family. For middleaged and older populations, we will be able to determine risk profiles for numerous late-onset diseases, preferably before the appearance of symptoms, which at least could be partly prevented through dietary or pharmaceutical interventions. In the near future, the monitoring of individual drug response profiles with DNA tests throughout life will be standard practice. Soon, genetic testing will comprise a wide

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spectrum of different analyses with a host of consequences for individuals and their families – an issue worth emphasizing when explaining genetic testing to the public. The challenge for health care professionals will be to correctly interpret the outcome of genetic testing for their patients, their patients families, and for society in general. Genetic counselors, who explain the purpose and results of genetic tests, will be crucial for helping individuals to make informed decisions, particularly when test results indicate the possibility of disease. Current training programs, including those in medical schools, do not adequately teach students how to deal with these challenges. The tremendous potential for efficient information transfer via the Internet can and should be used to inform the public of the possibilities provided by the genomics era. However, when it comes to sensitive and very personal aspects of genetic information, traditional contact with health care professionals is still the most appropriate route. Reaping the fruits of the human genome sequencing project through alleviating the suffering of patients will only be possible if available genetic information is combined with the skilled professionalism of health care workers and ethically solid standards. In the “post-genomic” era, the natural progression is towards interrogating the main effectors of physiological functions – proteins. The major genomic projects of the last decade have shaped proteome-wide sequencing, mapping, and analysis. For example, the creation of the Human Proteome Organization’s Human Brain Proteome Project to foster the effective international communication of brain related proteomic data. Complex diseases are now rapidly investigated by novel highthroughput biochemical inquires to uncover disease dynamics, clinical markers, and drug targets. Contrary to the genome, the proteome is composed of an active array of molecules constantly being modified and with special localization. Proteomic approaches are able to characterize post-translational modifications, a method by which the cell dynamically and quickly modifies protein function and regulates both creation and degradation in response to cellular perturbations (e.g., disease provocation). Protein profiling and identification techniques using mass spectrometry (MS) and bioinformatics can lead to the discovery, identification, and characterization of protein biomarkers differentially expressed in the diseased states versus the control.

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Chapter 22

Biochips

Abstract Biochip technology is highly effective method that allows monitoring of thousands of genes/alleles at a time in computerized automatic operations with minimal volumes of necessary reagents. Biochips promise an important shift in molecular biology, DNA diagnostics, and pharmacology, research in carcinogenesis and other diseases and also the possibility of a better understanding of the world of biology in its globality. Keywords Biochips · Microarray · Hybridization · Gene expression · DNA diagnostics · Peptide nucleic acid · Oligonucleotide · Protein arrays

22.1 Prologue Technological advances in miniaturization have found a niche in biology and signal the beginning of a new revolution. The biological and physical sciences share a common interest in small structures (the definition of “small” depends on the application, but can range from 1 nm to 1 mm). A vigorous trade across the borders of these areas of science is developing around new materials and tools (largely from the physical sciences) and new phenomena (largely from the biological sciences). The physical sciences offer tools for synthesis and fabrication of devices for measuring the characteristics of cells and sub-cellular components, materials useful in cell and molecular biology; biology offers a window into the most sophisticated collection of functional nanostructures that exists (Whitesides, 2003). Most of the attention and advances have been made with DNA chips yet a lot of progress is being made in the use of other biomolecules and cells. Development of high-throughput “biochip” technologies has dramatically enhanced our ability to study biology and explore the molecular basis of disease. Biochips enable massively parallel molecular analyses to be carried out in a miniaturized format with a very high throughput.

22.2 Concept A new revolution technology that may become promising for research, diagnostics and therapy enters into biology and medicine. Biochips containing microarrays M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_22,

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of genetic information promise to be important research tools in the post genomic era (Jain, 2001). The basic idea of the biochip technology is to convert the chemistry of life into a static form programmed to monitor genes, proteins and relations between them. Biochip programmed by known sequences of DNA/RNA or proteins can recognize the real genes, mutations and levels of expression. Biochips are collections of miniaturized test sites (microarrays) arranged on a solid substrate onto which a large number of biomolecules are attached with high density. The word “biochip” derives from the computer term “chip”. Although silicon surfaces bearing printed circuits can be used for DNA binding, the term biochip is now broadly used to describe all surfaces bearing microscopic spots, each one being formed by specific capture probes. The capture probes are chosen to complement the target sequence to be detected. Each capture probe will bind to its corresponding target sequence. Like a computer chip performing millions of mathematical operations in a few split seconds, a biochip allows for simultaneous analyses of thousands of biological reactions, such as decoding genes, in a few seconds. Biochip technologies can be applied to numerous fields including genomic, proteomic, and glycomic research, as well as pharmacology and toxicology. However, one of the most common applications is in the determination of gene expression in human cells and tissues. Global gene expression analysis has helped to identify important genes and signalling pathways in human malignant tumours. Biochips are formed by in situ (on chip) synthesis of oligonucleotides (Braun et al., 2005) or peptide nucleic acids (PNAs) or spotting of DNA fragments. Hybridisation of RNA- or DNA-derived samples on chips allows the monitoring of expression of mRNAs or the occurrence of polymorphisms in genomic DNA. Basic types of DNA chips are the sequencing chip, the expression chip and chips for comparative genomic hybridisation. Like a computer chip performing millions of mathematical operations in a few split seconds, a biochip allows simultaneous analyses of thousands of biological reactions, such as decoding genes, in a few seconds. Advanced technologies used in automated microarray production are photolithography, mechanical microspotting and ink jets. Bioelectronic microchips contain numerous electronically active microelectrodes with specific DNA capture probes linked to the electrodes through molecular wires. Several biosensors have been used in combination with biochips. The purpose of the chips is to detect many genes present in a sample in one assay rather than performing individual gene assays as is the practice e.g. in so-called multiwells, plates with 96 wells, where the reactions take place. The huge amount of information coming from the genome sequence and other research genome programs cannot be utilised to the full without the availability of methods such as biochips which enable these genes or specific DNA sequences to be detected in biological samples. DNA chip technology is an example of the enormous efforts undertaken in the genomic field in the last few years.

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22.3 Design of Biochip Use of protein array technology over conventional assay methods has advantages that include simultaneous detection of multiple analytes, reduction in sample and reagent volumes, and high output of test results. The susceptibility of ligands to denaturation, however, has impeded production of a stable, reproducible biochip platform, limiting most array assays to manual or, at most, semi automated processing techniques. Such limitations may be overcome by novel biochip fabrication procedures (FitzGerald et al., 2005). A design for a biochip memory device based on known materials and existing principles is presented by Robinson and Seeman, (1987). The fabrication of this memory system relies on the self-assembly of the nucleic acid junction system, which acts as the scaffolding for a molecular wire consisting of polyacetylene-like units. A molecular switch to control current is described which is based on the formation of a charge – transfer complex. A molecular-scale bit is presented which is based on oxidation – reduction potentials of metal atoms or clusters. The readable “bit” which can be made of these components has a volume of 3×107 Å3 and should operate at electronic speeds over short distances. After selection of a suitable biochip substrate, biochip surfaces are chemically modified and assessed to enable optimization of biochip fabrication procedures for different test panels. Each biochip has hundreds to thousands of gel drops on a glass, plastic or membrane support, each about 100 microns in diameter. A segment of a DNA strand, protein, peptide or antibody is inserted into each drop, tailoring it to recognize a specific biological agent or biochemical signature. These drops are in known positions so when a sample reacts, the reaction position can be detected, identifying the sample. The biochip system can identify infectious disease strains in less than 15 minutes when testing protein arrays and in less than two hours when testing nucleic acid arrays.

22.4 Applications of Biochips The complete sequence of the human genome and subsequent intensive searches for polymorphic variations are providing the prerequisite markers necessary to facilitate elucidation of the genetic variability in drug responses. Improvements in the sensitivity and precision of DNA microarrays permit a detailed and accurate scrutiny of the human genome. These advances have the potential to significantly improve health care management by improving disease diagnosis and targeting molecular therapy. Pharmacogenetic approaches, in limited use today, will become an integral part of therapeutic monitoring and health management, permitting patient stratification in advance of treatments, with the potential to eliminate adverse drug reactions (Hardiman, 2008). Biochip technologies can be applied to numerous fields including genomic, proteomic, and glycomic research, as well as pharmacology and toxicology (Fig. 22.1).

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Fig. 22.1 Biochips is used as a molecular diagnostic tool in point-of care diagnosis

However, one of the most common applications is in the determination of gene expression in human cells and tissues. Global gene expression analysis has helped to identify important genes and signaling pathways in human malignant tumors. And there is hope that microarrays will make the step from “the (laboratory) bench to the bedside (of the patient)”. Biochips (e.g., GeneChip, CYP450, electrochemical biochips, protein biochips, microfluidic biochips and nanotechnology-based biochips) are assuming an important role in molecular diagnostics, and their application in point-of-care diagnosis is expected to facilitate the development of personalized medicine. Gene expression profiling by microarrays should advance the progress of personalized cancer treatment based on the molecular classification of subtypes. Refinements in biochip miniaturization with the advent of nanotechnology will further contribute to molecular diagnostics and the development of personalized medicine.

22.4.1 Biochip and Clinical Medicine In the next years biochips will enter into clinical medicine. Development of highthroughput “biochip” technologies has dramatically enhanced our ability to study biology and explore the molecular basis of disease. Biochips enable massively parallel molecular analyses to be carried out in a miniaturized format with a very high throughput (Kallioniemi, 2001). Each larger diagnostics laboratory will offer genetic tests with this method. The technology provides a point-of-care diagnostic system that would save time and money compared to current systems, which require sending samples to a centralized lab for confirmatory diagnosis Jain (2004). Various biochip technologies in cancer research, including analysis of disease predisposition by using single-nucleotide polymorphism (SNP), global gene expression patterns by cDNA microarrays, concentrations, functional activities or interactions of proteins with proteomic biochips, and cell types or tissues as well as clinical endpoints associated with molecular targets by using tissue microarrays. One can predict that individual cancer risks can, in the future, be estimated accurately by a microarray profile of multiple SNPs in critical genes. Diagnostics of cancer will be facilitated by biochip readout of activity levels of thousands of genes and proteins. Biochip diagnostics coupled with informatics solutions will form the basis of individualized treatment decisions for cancer patients. The 12 tumor markers’ (TMs) biochip diagnostic (C12) system has been proven useful in some previous studies but its value

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for colorectal cancer (CRC) only was not systematically investigated (Chen et al., 2008). Biochip sensors have been used for the rapid and sensitive detection of viral disease (Livingston et al., 2005). The biochip could be a feasible tool for rapidly diagnosing mastitis-causing pathogens in milk and providing information for a more effective treatment to cure mastitis. The biochip can detect 7 common species of mastitis-causing pathogens, including Corynebacterium bovis, Mycoplasma bovis, Staphylococcus aureus, and the Streptococcus spp. S. agalactiae, S. bovis, S. dysgalactiae, and S. uberis, within 6 h. This technique is based on DNA amplification of genes specific to the target pathogens and consists of 4 basic steps: DNA extraction of bacteria, polymerase chain reaction, DNA hybridization, and colorimetric reaction.

22.4.2 Biochips and Biosensors In the past two decades, the biological and medical fields have seen great advances in the development of biosensors and biochips capable of characterizing and quantifying biomolecules. Biochips (eg. Gene Chip, CYP450, electrochemical biochips, protein biochips, microfluidic biochips and nanotechnology-based biochips) are assuming an important role in molecular diagnostics. Their application in pointof-care diagnostics and enable parallel molecular analyses in a miniaturized format with a very high throughput. Refinements in biochip miniaturization with the advent of nanotechnology will further contribute to molecular diagnostics and the development of personalized medicine. Biochip platforms on gold can be manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH2 groups to N-hydroxysuccinimide (NHS) – activated surfaces. The potential application of tumor necrosis factor – (TNFα ) and serodiagnosis of hemophagocytic lymphohistiocytosis (HLH) has been investigated. Interactions of TNFα antigen and TNFα antibody on the biochips have been optimized using an indirect immunofluorescence method (Weidong et al., 2008).

22.4.3 Biochip and Pharmacogenetics Large-scale population researches, diagnostics of genetic predisposition to multifactorial diseases, screening of the polymorphic loci associated with individual sensitivity to pharmaceutical preparations require the development of effective, exact and rapid methods of analysis for detection of many mutations simultaneously. One of the most perspective methods to solve these problems is a method of allele-specific hybridization with biochips. Taking the analysis of mutations in genes CYP1A1, CYP2D6, GSTT1, NAT2, CYP2C9, CYP2C19 and MTHFR as an example, efficiency of using the approach for identification of individual genetic polymorphism can be studied. Biochips can also be a convenient tool in pharmacogenetics researchs.

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22.4.4 Biochip and Biowarfare The biochip can also be used to identify biological warfare agents. Easy sample preparation, standard operating protocols and a portable biochip reader that is smaller than a lunchbox make the system suitable for use in the field by first responders, military personnel and medical technicians. The system makes use of the polymerase chain reaction (PCR), a universal method for replicating billions of copies from one piece of genetic material. PCR allows trace quantities of DNA to be replicated to a level where they can be detected in the biochip system. A sample to be tested is applied to a biochip, which is then put in a reader and scanned using patented side illumination laser technology to detect reaction sites. Automated algorithms determine the agents present in the sample.

22.4.5 Biochip and Diagnostics Biochip can be used for rapid diagnostic testing since the biochip allows technicians to test for so many different agents at once. The technology holds great promise for rapid diagnostic testing since the biochip allows technicians to test for so many different agents at once. Under current development is a respiratory syndrome chip that tests for strep throat, influenza A, and influenza B (Lee et al., 2008). This chip will allow doctors to make a faster and more precise diagnosis when patients display symptoms common to several different ailments. The TBBiochip oligonucleotide microarray system is a rapid system to detect mutations associated with rifampin (RIF) resistance in mycobacteria (Caoili, et al., 2006). The lack of a sensitive immunoassay for quantitating serum prostate-specific membrane antigen (PSMA) hinders its clinical utility as a diagnostic/prognostic biomarker. An innovative protein biochip immunoassay was used to quantitate and compare serum PSMA levels in healthy men and patients with either benign or malignant prostate disease. These initial results suggest that serum PSMA may be a more effective biomarker than prostate-specific antigen for differentiating benign from malignant prostate disease and warrants additional evaluation of the surface-enhanced laser desorption/ionization PSMA immunoassay to determine its diagnostic utility (Xiao et al., 2001). An antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding (Blank et al., 2003). Technological advances in miniaturization have found a niche in biology and signal the beginning of a new revolution. Most of the attention and advances have been made with DNA chips yet a lot of progress is being made in the use of other biomolecules and cells. A variety of reviews have covered only different aspects

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and technologies but leading to the shared terminology of “biochips.” (Ng and Ilag, 2003).

22.4.6 Biochip and Cancer The various biochip technologies in cancer research, include analysis of (1) disease predisposition by using single-nucleotide polymorphism (SNP) microarrays, (2) global gene expression patterns by cDNA microarrays, (3) concentrations, functional activities or interactions of proteins with proteomic biochips, and (4) cell types or tissues as well as clinical endpoints associated with molecular targets by using tissue microarrays. One can predict that individual cancer risks can, in the future, be estimated accurately by a microarray profile of multiple SNPs in critical genes. The biochip system also has great potential as a discovery tool. Current research aimed at the development of proteome chips has shown great promise. Proteome chips, which are biochips displaying all the proteins expressed by an organism at a specific time, provide the ability to screen for new cancer biomarkers, vaccine targets and therapeutic targets, as well as provide a means of characterizing disease states. Comparative genomic hybridization (CGH) to metaphase chromosomes has been widely used for the genome-wide screening of genomic imbalances in tumour cells. Substitution of the chromosome targets by a matrix consisting of an ordered set of defined nucleic acid target sequences would greatly enhance the resolution and simplify the analysis procedure, both of which are prerequisites for a broad application of CGH as a diagnostic tool. However, hybridization of whole genomic human DNA to immobilized single-copy DNA fragments with complexities below the megabase pair level has been hampered by the low probability of specific binding because of the high probe complexity. High-copy-number amplifications contained in tumour cells are rapidly scored by use of target DNAs as small as a cosmid. Low-copy-number gains and losses are identified reliably by their ratios by use of chromosome-specific DNA libraries or genomic fragments as small as 75 kb cloned in PI or PAC vectors as targets, thus greatly improving the resolution achievable by chromosomal CGH. The ratios obtained for the same chromosomal imbalance by matrix CGH and by chromosomal CGH corresponded very well. The new matrix CGH protocol provides a basis for the development of automated diagnostic procedures with biochips designed to meet clinical needs (Solinas-Toldo et al., 1997).

22.4.7 Biochip Department of Defense Development of an implantable biochip that could relay vital health information if a soldier is wounded in battle or a civilian is hurt in an accident has been done. The Department of Defense has awarded $1.6 million to the Center for Bioelectronics,

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Biosensors and Biochips (C3B) at Clemson University for the development of an implantable biochip that could relay vital health information if a soldier is wounded in battle or a civilian is hurt in an accident. The biochip, about the size of a grain of rice, could measure and relay such information as lactate and glucose levels in the event of a major hemorrhage, whether on the battlefield, at home or on the highway. Biosensors are also widely used for biowarfare detection, including anthrax, for the Department of Defense. Biochip has other long-term potential applications, such as monitoring astronauts’ vital signs during long-duration space flights and reading blood-sugar levels for diabetics.

22.5 Biochip Market During the past ten years, major pharmaceutical (“pharma”) companies have seen their R&D costs explode as their actual productivity has declined. Drug companies have looked outside of their labs to find drug products faster. As a result, pharma companies have increasingly become motivated research and financial partners with many biotechnology companies. At the same time, pharma companies also want to become customers and partners of biochip companies because these companies have technologies that might help them become more productive faster. The products from the biochip market help to accelerate the research processes and capabilities of bio-pharmaceutical drug discovery and basic academic bioresearch. New business models are being created. The ideas of pharmacogenomics and targeted therapies have received positive reception from government regulators. Roche and Affymetrix were the first companies to receive FDA approval for a biochip-based, molecular diagnostic test and instrument system. Basic academic research is helping as well. Accelerating the drug making process requires updated industrialization of R&D, done through the use of genomics and proteomics technologies such as biochips that the biotechnology companies provide.

22.6 Future of Biochips Differentiation and prognosis in breast cancer and individualized drug therapy (pharmacogenetics) are the two potential applications of biochips (Gary, 2008). In cancer the gene expression profile permits an accuracy of differentiation and prognosis that was impossible so far. Soon oncologists will make their therapeutic decisions on the basis of biochip-based gene expression profiles. For an individualized drug therapy extensive genetic tests must be accomplished, above all of the cytochrome P450 system and an already available biochip seems to make this possible reliably and economically. Moreover, in science biochips will play a substantial role in the analysis of the genetic basis of common diseases. If these diseases are understood once, the clinical use of biochips will open an enormous potential for a predictive medicine. DNA chips will facilitate the integration of diagnosis and therapeutics, as well as the introduction of personalized medicines.

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In recent years (Zhang et al., 2008) the application of gold nanoparticles instead of fluorescence dyes and enzyme-conjugation in biochips is very common. For example, Au nanoparticles labelling method was applied in many DNA-detection methods, and a novel readout scheme for gold nanoparticle-based DNA microarrays was studied relying on “Laser-Induced Scattering around a nanoAbsorber” and nanogold electrode, and the colorimetric detection using gold label plus silver stain was also developed. The quality analysis of probe spots can be obtained by using gold nanoparticles with positive charges to label DNA through electrostatic attraction. The probe spots can also be detected by a simple personal computer scanner. Gold nanoparticles deposited on a glass surface can be dissolved in brominebromide solution. The same microarray treated with gold particles staining and destaining can still be used for hybridization with nearly the same efficiency (Hsiao and Chen, 2009). The technology is a good combination of gene technology and nanotechnology. At the same time, a number of scientists from different countries have paid more attention to the application of nanoparticles in biochips and have some new patents recieved. This approach makes quality control of a microarray chip feasible and should be a valuable tool for biomarker discovery in the future. Protein arrays are emerging to follow DNA chips as possible screening tools. Array-based protein technologies are emerging for basic biological research, molecular diagnostics and therapeutic development with the potential of providing parallel functional analysis of hundreds or perhaps hundreds of thousands of proteins simultaneously. Array-based methods are becoming prevalent within proteomics research due to the desire to analyze proteins in an analogous format to that of the DNA microarray. Novel protein biochips are under development in academic laboratories and emerging biotechnology companies to advance the pace and scope of scientific discovery. Biochips are a relevant topic for insurers partially already today, much more however in the future.

References Blank, K., Mai, T., Gilbert, I., Schiffmann, S., Rankl, J., Zivin, R., Tackney, C., Nicolaus, T., Spinnler, K., Oesterhelt, F., Benoit, M., Clausen-Schaumann, H., Gaub, H.E., 2003, A forcebased protein biochip. Proc Natl Acad Sci USA 100:11356–11360. Braun, Y., Wai, D.H., Biemel, K., Schäfer, K.L., Gabbert, H.E., Poremba, C., 2005, Biochip analysis: status quo. Verh Dtsch Ges Pathol 89:116–124. Caoili, J.C., Mayorova, A., Sikes, D., Hickman, L., Plikaytis, B.B., Shinnick, T.M., 2006, Evaluation of the TB-biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis. J Clin Microbiol 44:2378–2381. Chen, C., Chen, L.Q., Yang, G.L., Li, Y., 2008, The application of C12 biochip in the diagnosis and monitoring of colorectal cancer: systematic evaluation and suggestion for improvement. Clin Rep 54:186–190. Fitzgerald, S.P., Lamont, J.V., McConnell, R.I., Benchikh, el, O., 2005, Development of a high-throughput automated analyzer using biochip array technology. Clin Chem 51: 1165–1176. Gary, H., 2008, Applications of microarrays and biochips in pharmacogenomics pharmacogenomics in drug discovery and development: from bench to bedside. Methods Mol Biol 448:21–30.

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Hardiman, G., 2008, Applications of microarrays and biochips in pharmacogenomics. Methods Mol Biol 448:21–30. Hsiao, C.R., Chen, C.H., 2009, Characterization of DNA chips by nanogold staining. Anal Biochem 389:118–123. Jain, K.K., 2001, Biochips for gene spotting. Science 294:621–623. Jain, K.K., 2004, Applications of biochips: from diagnostics to personalized medicine. Curr Opin Drug Discov Devel 7:285–289. Kallioniemi, O.P., 2001, Biochip technologies in cancer research. Ann Med 33:142–147. Lee, K.H., Lee, J.W., Wang, S.W., Liu, L.Y., Lee, M.F., Chuang, S.T., Shy, Y.M., Chang, C.L., Wu, M.C., Chi, C.H., 2008, Development of a novel biochip for rapid multiplex detection of seven mastitis-causing pathogens in bovine milk samples. J Vet Diagn Invest 20:463–471. Livingston, A.D., Campbell, C.J., Wagner, E.K., Ghazal, P., 2005, Biochip sensors for the rapid and sensitive detection of viral disease. Genome Biol 6:112. Ng, J.H., Ilag, L.L., 2003, Biochips beyond DNA: technologies and applications. Biotechnol Annu Rev 9:1. Robinson, B.H., Seeman, N.C., 1987, The design of a biochip: a self-assembling molecular-scale memory device. Protein Eng 1:295–300. Solinas-Toldo, S., Lampel, S., Stilgenbauer, S., Nickolenko, J., Benner, A., Döhner, H., Cremer, T., Lichter, P., 1997, Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances. Genes Chrom Cancer 20:399–407. Weidong, Du., Xueling, Ma., Schneider, E.M., 2008, A direct immunoassay assessment of streptavidin- and n-hydroxysuccinimide-modified biochips in validation of serological TNF responses in hemophagocytic lymphohistiocytosis. J Biomol Screening 13:515–526. Whitesides, G.M., 2003, The ‘right’ size in nanobiotechnology. Nat Biotechnol 21:1161–1165. Xiao, Z., Adam, B.-L., Cazares, L.H., Clements, M.A., Davis, J.W., Schellhammer, P.F., Dalmasso, E.A., Wright, G.L. Jr., 2001, Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease. Cancer Res 61:6029–6033. Zhang, Y.Q., Wang, Y.F., Jiang, X.D., 2008, The application of nanoparticles in biochips. Recent Pat Biotechnol 2:55–59.

Chapter 23

Personalised Medicine

Abstract Towards the post genomic sequencing era, conventional drug discovery is drastically improving genomic technologies and computational advances. One of the potential benefits is the accelerated introduction of new diagnostics and treatments to the general public. Personalized medicine is typically described as the use of molecular or genetic characteristics to customize therapy. The right medication for the right patient is the goal of personalized medicine, which directly benefits from many of biotechnology’s biggest and most recent advances. Personalized medicine approach is increasingly incorporated into health care, understanding patients’ needs and their readiness to adopt these novel technologies will become progressively more important for the development of appropriate health policies. Keywords Pharmacogenomics · Drug · Predisposition · Personalised medicine · Drug discovery · Pharmaceutical industry · Genetic markers · Cytochrome P450 · Patent · Ethical legal social issues

23.1 Prologue With the beginning of the post-genome era, the promise of tailoring the practice of medicine to the individual has started. The completion of the entire genome sequence of many experimental organisms as well as the human allow us to compare several genomic sequences, to get valuable information for gene discovery and functional genomics. Driven by chemistry but increasingly guided by pharmacology and the clinical sciences, drug research has contributed more to the progress of medicine during the past century than any other scientific factor. The advent of molecular biology and, in particular, of genomic sciences is having a deep impact on drug discovery. Recombinant proteins and monoclonal antibodies have greatly enriched our therapeutic armamentarium. Genome sciences, combined with bioinformatics tools, allow us to dissect the genetic basis of multifactor diseases and to determine the most suitable points of attack for future medicines, thereby increasing the number of treatment options (Priori et al., 1999). The dramatic increase in the complexity of drug research is enforcing changes in the institutional basis of M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_23,

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this interdisciplinary endeavour. The biotechnology industry is establishing itself as the discovery arm of the pharmaceutical industry. In bridging the gap between academia and large pharmaceutical companies, the biotech firms have been effective instruments of technology transfer. Molecular, pharmacological and patient clinical data will be captured at various phases and integrated in a “knowledge management system” that will be used to facilitate rational drug design around molecular diseases.

23.2 Concept In recent years, tools for the development of new drugs have been dramatically improved. These include genomic and proteomic research, numerous biophysical methods, combinatorial chemistry and screening technologies (Schwardt et al., 2003). As new therapeutic treatments emerge, molecular diagnostics will increasingly spin off products from drug development data to compliment the therapies allowing a more and more targeted approach to drug selection. Initial applications of molecular diagnostics were mostly for infections but are now increasing in the areas of genetic disorders, preimplantation screening and cancer. Genetic screening tests, despite some restrictions are a promising area for future expansion of in vitro diagnostic market. Molecular diagnostics is being combined with therapeutics and forms an important component of integrated healthcare. Molecular diagnostic technologies are also involved in development of personalized medicine based on pharmacogenetics and pharmacogenomics. Currently, there has been a considerable interest in developing rapid diagnostic methods for point-of-care and biowarfare agents such as anthrax. Personalized medicine simply means the prescription of specific therapeutics best suited for an individual based on pharmacogenetic and pharmacogenomic information (Jain, 2002). Several technologies are used including single nucleotide polymorphism genotyping, haplotyping, gene expression studies by biochip/microarrays and proteomics. Molecular diagnostics will play an important role in the development of personalized medicine, in which therapy and diagnosis will be integrated. There are several examples of the personalized medical approach, which include genotype-based selection of patients for effective cancer therapy, to spare those who would not respond or would suffer undesirable side effects. This newly emerging field uses the patient’s genetic composition to tailor strategies for patient-specific disease detection, treatment, or prevention. The regulations governing personalized medicine can be complicated because they encompass in vitro diagnostic systems and laboratory tests as well as methods of disease treatment and patient care (Richmond, 2008). Personalized therapy is financially feasible, as it will reduce the costs of drug development by shortening the drug development cycle. The introduction of pharmacogenomics into clinical trials is reducing the chances of failed clinical trials and increasing the prospects of safer and more effective therapies for specific

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groups of patients. Several advantages, as well as challenges to the development of personalized medicine are examined. Personalized medicine is anticipated to be an acceptable part of medical practice by the year 2010 (Jain, 2002). Personalization of medicine requires two characterizations: a well-grounded understanding of who the patient is and an equally robust understanding of the subpopulation that most resembles that patient in the context of the decisions at hand. These characterizations are readily represented and can be used to drive decision-making in a rational manner that maximizes the positive outcomes for the patient (Kohane, 2009). Not only is personalized medicine tailoring the right drug, for the right person, at the right time but it also includes evaluating predisposition to disease sometimes decades in advance of its threatened onset (Ginsburg and McCarthy, 2001). The key task is to find genes and gene variations that play a role in a disease. The first step is to associate the occurrence of a particular gene variant with the incidence of a particular disease or disease predisposition – an association that can vary from one individual to another depending on many factors, including environmental circumstances. The outcome is the development of biomarkers which are stable and predictive. Today’s biomarker is tomorrow’s theranostic. Personalized medicine will provide the link between an individual’s molecular and clinical profiles, allowing physicians to make the right patient-care decisions and allowing patients the opportunity to make informed and directed lifestyle decisions for their future well-being. Molecular diagnostics, the use of DNA-, proteinor mRNA-based biological markers to predict the risk of developing disease or the molecular phenotype of an existing one, will change the way we currently define disease. Genomic analysis of diseases with homogeneous clinical phenotypes will unveil distinct molecular entities that require different treatment strategies for optimal outcomes. Clinical diseases as we know them will be replaced by molecular classification. Therapies directed at the root cause of disease will replace those that simply treat the symptoms of disease. Finally, a pharmacogenomic test that predicts therapy response based on a patient’s genomic profile will accompany many drugs. Personalized medicine will involve radical changes in the pharmaceutical industry and medical practice and is likely to affect many aspects of society. Most importantly, the individual whose health is at stake will benefit enormously. The promise of personalised medicines tailored to a person’s genes has been “overhyped”, particularly for developing countries. Personalized diagnostic tests are used to detect patient-to-patient variations in gene or protein expression levels, which act as indicators for drug treatments or disease prognosis. The aim of personalized medicine or individualized treatment is to match the right drug to the right patient and, in some cases, even to design the appropriate treatment for a patient according to his/her genotype. Basic technologies of molecular diagnostics play an important role, particularly those for single nucleotide polymorphism (SNP) genotyping. Diagnosis is integrated with therapy for selection of the treatment as well for monitoring the results. Biochip/microarray technologies are also important and finally bioinformatics is needed to analyze the immense amount of data generated by various technologies.

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23.3 Practice of Medicine in the Twenty-First Century In the next decade, medical care will undergo revolutionary changes. No longer will medical practice be limited to the empirical extrapolation of a patient’s care from generalized clinical-trial results. Traditional medical practice, based on trialand-error, results in both under-treatment and over-treatment, multiple office visits, the need for drug monitoring, and frequent regimen changes. More than 100,000 deaths per year are attributed to adverse drug (Lazarou et al., 1998). A personalized approach of tailored care for every individual based on their specific, molecular disease will become the standard of care. In the prototypical office visit of 2015, the physician will examine a patient’s genetic profile (stored on CD ROMs or equivalent), lifestyle, and results from objective molecular screening and monitoring tests. Algorithms, derived from previous research efforts, will be used to compute the likelihood that a patient develops a host of chronic diseases. The focus of medicine at this juncture will be entirely preventive. Lifestyle modifications and the use of prophylactic therapy will be recommended based on what is best for that patient to avoid chronic disease to which they could be susceptible. The “office” of the future might be virtual; internet office visits might supplement some of the direct patient–physician contact Issa et al. (2009). Patients will be more knowledgeable of their own health and risk profiles and more active in directing their own healthcare (Triggle, 2007).

23.4 Role of Personalized Medicine in Drug Discovery The personalized medicine strategy for drug discovery and development should yield a spectrum of product opportunities for the pharmaceutical industry. Diagnostic risk assessment and disease-monitoring tools that accurately quantify disease burden in patients will be a direct outcome of research during the early discovery process. Pharmacogenomic markers of efficacy and side effects will be used in conjunction with specific drugs to target drug therapy to those patients who will have an optimal response (Sadee, 2002). The business rationale for targeted therapies, which some argue will decrease market share, is that such products will eventually expand the market by recruiting patients from less effective therapies or by identifying less symptomatic individuals who might benefit from prophylactic therapy (Fig. 23.1). The clinical phases of drug development (Fig. 23.2) afford the opportunity to capture patient clinical data, imaging and in vitro molecular response data simultaneously. Academic medical centres and clinical research organizations are now conducting clinical trials with future research in mind. Archiving biological specimens along with traditional clinical covariates is becoming routine. Some centres are also actively engaged in pharmacogenomic marker research. In the near future, clinical trials might be conducted in specialized units where detailed clinical, biological and genomic data are collected and integrated. Genome- and proteomewide profiles together with biological pathway databases, imaging and clinical data

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Fig. 23.1 The role of molecular diagnostics in the development of personalised medicine testing

Fig. 23.2 Different phases of new drug discovery

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on every patient will be used to analyze an individual’s disease and drug response (Baba, 2001). The understanding of the biology of disease and drug action gleaned from these sophisticated new paradigms will dramatically accelerate the realization of truly personalized medicine. Molecular pathology is in a state of rapid evolution featuring continuous technology developments and new clinical opportunities for drug selection, predicting efficacy and toxicity, and monitoring disease outcome. The approvals of trastuzumab (Herceptin, Genentech, South San Francisco, CA) for the treatment of HER-2/neu overexpressing breast cancer and imatinib (Gleevec, Novartis Pharmaceuticals, East Hanover, NJ) for the treatment of chronic myelogenous leukemia featuring a bcr/abl translocation and gastrointestinal stromal tumors with selective c-kit oncogeneactivating mutations have brought to the diagnostic laboratory an expanding role for the testing of patients to determine their eligibility to receive these new therapies.The introduction of targeted therapeutics into clinical practice has created major opportunities for further development of the molecular pathology. A variety of new approaches to drug use have introduced genetically prescribed medications. Moreover, modern medicine now is driven by rapid communication and electronic information sharing, which has created more informed and demanding consumers. The advantages of personalized medicine to the pharmaceutical industry are as follows: • • • •

Increased efficiency and reduced costs of target and lead discovery Reduced timelines and costs of clinical trials Emergence of new gene targets for drug discovery Product differentiation in the market place.

Personalized medicine offers many advantages to patients and clinician like higher probability of desired outcome with a drug, low probability of untoward side effects, preventive strategies, focused therapies, reduced costs and better health and better healthcare. Biological therapies such as those which use patient’s own cells are considered to be personalized medicines. Vaccines are prepared from individual patient’s tumor cells. Individualized therapeutic strategies using monoclonal bodies can be directed at specific genetic and immunologic targets. Ex vivo gene therapy involves the genetic modification of the patient’s cells in vitro, prior to reimplantation of these cells in the patient’s body. Various technologies are integrated to develop personalized therapies for specific therapeutic areas described in the report. Examples of this are genotyping for drug resistance in HIV infection, personalized therapy of cancer (Veer and Bernand, 2008), antipsychotics for schizophrenia, antidepressant therapy, antihypertensive therapy and personalized approach to neurological disorders. Although genotyping is not yet a part of clinically accepted routine, it is expected to have this status by the year 2010. Several players are involved in the development of personalized therapy. Pharmaceutical and biotechnology companies have taken a leading role in this venture in keeping with their future role as healthcare enterprises rather than mere developers of technologies and manufacturers of medicines.

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Molecular Diagnosis Will Determine Prognosis and Therapy

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Drug discovery is a continuum of the processes, i.e. identification of diseaseassociated targets, screening of compound libraries, identification of leads and their optimization to drugs, preclinical and clinical drug development. Recent advances in genomic sciences will greatly reinforce processes such as early target identification and clinical researches. Particularly, exploitation of genomic information in clinical researches is expected to revolutionarily improve drug development and eventual clinical use. Ultimately, tailor-made medicine will provide us with great benefit in the near future therapy (Nohmi, 2002). Genetic heterogeneity appears to be a significant source of variability observed in the response to drugs. This variability means that information pertaining to interethnic and interindividual genetic differences can be used to facilitate rational drug discovery and development and to avoid or minimize the incidence of adverse events in clinical trials. Thus, one could generate criteria for selecting patients most likely to benefit from a drug without incurring unnecessary risk. Early or preventive therapy guided by genotyping could significantly enhance clinical outcome. The need for a new, individualized approach to drug development and therapy is clear. Every year, approximately 3.1 billion prescriptions are issued in the United States, of which approximately 2.1 million results in an adverse reaction. One million prescriptions from this latter group may result in hospitalization, and of these more than 100,000 patients may die. How can we reduce these severe adverse reactions by using pharmacogenomics? Over the near term, patient genotyping prior to therapy in a few but increasing number of instances will serve to avert or minimize severe drug toxicity. Alternatively, drugs may be designed a priori so that they are not subject to the differential metabolic patterns known to be caused by polymorphic variation. Looking farther ahead, and on a much broader scale, the efficacy of administered drugs may be improved, rather than avoiding toxicity as the main objective, by distinguishing good responders from poor responders prior to therapy. Often, effective drug response is limited to a portion of treated patients, whereas the majority benefits little or not at all. Predicting which patients are most likely to respond best to a particular drug, or which drug will yield optimal effects for a given patient, would represent a significant advance in therapy even with current drugs (Schwardt et al., 2003), let alone novel drugs developed with these criteria in mind. The success of this approach depends in large part on assembling an extensive, high-quality database of informative SNPs, a major focus for genomics companies. Ultimately the vision of pharmacogenomics encompasses a genetic profile for each individual, containing sufficient information to select which drugs are most likely to be safe and effective in that person. The same insight will serve to prevent disease to begin with, arguably the most desirable goal.

23.5 Molecular Diagnosis Will Determine Prognosis and Therapy Personalized medicine is rooted in the hypothesis that diseases are heterogeneous, from their causes to rates of progression to their response to drugs. Each person’s

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disease might be unique and therefore that person needs to be treated as an individual. With limited understanding of the molecular basis of disease, we have relied on non-specific clinical signs. As genomic tools are sharpened, so will be our ability to dissect disease into its component parts (Nohmi, 2002). Clinical phenotypes thought to be one disease will be subclassified by a new genomic taxonomy. Recent discoveries in the molecular pathology of cancer have highlighted important and clinically significant differences in the gene expression patterns of a variety of tumors, including leukemias and breast cancer (Golub et al., 1999). In cardiovascular disease, genetic heterogeneity has been identified in the Long QT syndrome, a disorder of ventricular depolarization where clinical manifestations range from no visible signs to sudden death (Zareba et al., 1998). The etiology of the Long QT syndrome is attributed to mutations in one of at least four different ion channels (HERG, KVLQT1, SCN5A or KCNE1). The clinical course of the disease, level of aggressive therapy and choice of therapy (Na+ -channel blocker versus K+ -channel blocker versus beta blocker) are now determined by the genetic etiology (Shimizu and Antzelevitch, 1997; Schwartz et al., 1995) of the syndrome. Familial hypertrophic cardiomyopathy is another example of a genetically heterogeneous disease with a clinical phenotype of ventricular hypertrophy (Fananapazir, 1999). Familial cardiomyopathy results from >80 different mutations, each affecting the expression of a cardiac muscle sarcomeric protein. Mutationspecific prognoses have been established that mandate screening to determine who requires more frequent clinical monitoring, therapeutic intervention and family screening. As the underlying molecular architecture of other diseases is determined, medical practice will be tailored to properly diagnose and treat them.

23.6 Personalized Medicine and Genetic Markers The past few years have seen major advances in technology and the growth of genomic information as a byproduct of the human genome project. As a result, new and innovative markers of disease are being uncovered at an unprecedented rate. At the DNA level, >350 genetic tests are currently available (http://www.genetests.org). Although most tests are for rare, monogenic disorders, some are becoming available for more common, complex diseases. Examples include ApoE testing among dementia patients for differential diagnosis of Alzheimer disease (Eldik et al., 2002), and Factor V Leiden testing for predisposition to venous thrombosis. Furthermore, for most common diseases, a large number of genetic markers suggesting association with disease are reported in the literature. Advances in SNP-discovery technologies are providing opportunities for large-scale candidate gene studies (Crouch, 2001; Cargill et al., 1999). Indeed, whole-genome association studies are being contemplated for finding genetic predisposition markers for common, complex diseases. The next three to five years will see an explosion of new information in this area and the development of new predictive tests for complex diseases. Identification of RNA and protein markers for screening, diagnosis, prognosis and monitoring is also under way, facilitated through advances in transcript profiling

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Challenges of Realizing the Promise of Personalized Medicine

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and proteomics. The basic research methods used in the discovery of these markers require access to relevant disease tissues. Because tumor samples are routinely biopsied or removed, the first disease area likely to benefit from these technologies is cancer. Advances in the ability to classify disease are best illustrated in the work of Golub et al., 1999). Expression patterns (Hedenfalk et al., 2001; Waring et al., 2001) of 50 genes were determined to distinguish accurately between acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Similar approaches have been taken to identify candidate prognostic markers for melanoma. Clark et al. (2000) and Bittner et al. (2000) used transcript profiling to compare metastatic with nonmetastatic human melanoma cell lines. As a result, they identified several genes that are selectively unregulated in the metastatic lines that could have use in patient management. The acknowledgement that post-transcriptional modification of proteins might be an important determinant of disease is one factor driving the use of proteomic technologies for discovering molecular disease markers. These technologies include traditional 2D gel electrophoresis in addition to more advanced mass-spectrometry methods. Proteomic analysis might be used in medical microbiology in which the entire proteome of an organism can be studied or in diseases such as rheumatoid arthritis or central nervous system disorders in which protein-rich fluids at the site of injury, such as synovial joint and cerebral spinal fluid, respectively, are available for analysis. Cell line supernatants or explants from tumor tissues have already been used in large-scale expression profiling experiments to identify cancer markers. Page et al. (1999) compared the proteome of purified normal human luminal (from which most breast cancer is derived) with that of myoepithelial breast cells. They detected 170 proteins that differed between the two cell types. These experiments might shed light onto the process of cancer development and ultimately find use as cancer diagnosis or monitoring markers. Pharmacogenomic tests are finding their way into practice in several disease areas. Genotype resistance testing of HIV isolates has demonstrated clinical utility and provides a way to assist therapeutic decision-making in patients whose levels of HIV RNA are rising (Durant et al., 1999). In addition, assays are available to detect the HER2 protein receptor or copies of the HER2 gene sequence to determine eligibility for herceptin treatment or Adriamycin treatment, respectively, in node-positive breast cancer patients (Tsongalis et al., 2000). The Food and Drug Administration (FDA) approval of the pharmacogenomic marker HER2 linked to herceptin represents an important precedent for regulatory approval of personalized medicine products.

23.7 Challenges of Realizing the Promise of Personalized Medicine It is widely anticipated that during the next 5 years the molecular diagnostic industry will continue to grow at double-digit pace to meet increasing demand for personalized medicine. A wide variety of drugs in late preclinical and early

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clinical development are being targeted to disease-specific gene and protein defects that will require co-approval of diagnostic and therapeutic products by regulatory agencies. An increasingly educated public will demand more information about their predisposition for serious diseases and how these potential illnesses can be detected in an early stage when they can be arrested or cured with new therapies custom-designed for their individual clinical status. To respond to this demand, major pharmaceutical companies will partner with diagnostics companies or develop their own in-house capabilities that will permit efficient production of more effective and less toxic integrated personalized medicine drug and test products. For clinical laboratories and pathologists, this integration of diagnostics and therapeutics represents a major new opportunity to emerge as leaders of the new medicine, guiding the selection, dosage, route of administration, and multidrug combinations and producing increased efficacy and reduced toxicity of pharmaceutical products. In spite of the achievement of a complete human genome sequence, there are numerous challenges in realizing the vision of personalized medicine. Identifying genetic variants that are markers of disease or drug response requires sifting through several million SNPs in the human genome to find those that contribute to the disease and then demonstrating that the SNPs are clinically valid markers and are useful for managing patients (Halushka et al., 1999). To uncover DNA variants that predict common, complex diseases that result from a combination of genes and environmental factors will require cost-effective, high-throughput genotyping; large, well-characterized patient populations; sophisticated computational methodologies; and a detailed understanding of the biological pathways of disease (Fig. 23.3). Uncovering mRNA and protein markers for use in screening, diagnosis,

Fig. 23.3 The promise of personalized medicine

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Personalised Medicine and Pharmacogenomics

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prognosis and monitoring of disease will have its own set of challenges. Access to optimal relevant tissues might not be possible for many diseases. Proteomic technologies require further development, as do computational approaches for analyzing massive amounts of gene and protein profile data. To realize the vision of personalized medicine, the agenda for medical and pharmaceutical research must include the assembly and integration data from many sources on large numbers of patients. Clinical investigations should incorporate genotyping and molecular profiling technologies along with traditional clinical data collection and should establish a repository of patient samples where possible. New molecular markers might face many hurdles before they can be implemented in patient care. The issues range from FDA regulation and acceptance of these new markers, to developing tractable assay platforms, to resolving issues around the ethical, legal and social implications of obtaining highly sensitive genetic information. Foremost among these, in our opinion, is the education and engagement of physicians and patients in the paradigm shift to objective, quantitative marker-based clinical care. If appropriate patient management systems, integrated databases, educational tools and genetic counselling are not in place, then it will be difficult to realize the significant benefits forecast from this approach. Fortunately, we have already learned valuable lessons from past efforts to implement genetic screening for sickle cell anaemia, and from more recent efforts to screen for BRCA1 mutations in breast cancer families. The ethical, legal and social implications (ELSI) of human genetic research are the subject of a government-funded program (http://www.nhgri.nih.gov/ELSI/). The US government is playing an active role in addressing public concern over genetic information by, among other things, drafting legislation to protect patients from discrimination by employers and insurance companies. Furthermore, health professionals are rising to the challenge of educating both their members and the public. The American Medical Association has co-founded the National Coalition for Health Professional Education in Genetics (http://www.nchpeg.org) to promote health professional education and access to genetic information.

23.8 Personalised Medicine and Pharmacogenomics Personalized medicine and pharmacogenomics are inextricably linked. Pharmacogenomics is the use of genetic variations (such as SNPs, gene expression variability, or other molecular signatures) to understand and correlate with differential response to pharmaceutical agents (drugs). Pharmacogenomics can be deployed clinically to stratify patients into responders and non-responders and this practice is termed personalized medicine. To frame the context of current approaches, pharmacogenomics seeks to identify and validate the signature(s) of molecular analytes and these are converted to assays using the tools of molecular diagnostics. The deployment of these molecular diagnostics assays on defining and targeting patient populations is the domain of personalized medicine. This study includes all of the generally accepted imaging activities that are currently used in personalized

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medicine, including pharmacogenomics, genomics and theranostics. It examines associated clinical market segments in which personalized medicine has taken a prominent role including cancer treatment, cardiology and neurology markets. Medicine, as we move into the third millennium, still targets therapy to the broadest patient population that might possibly benefit from it, and it relies on statistical analysis of this population’s response for predicting therapeutic outcome in individual patients. Therapists of necessity make decisions about the choice of drug and appropriate dosage based on information derived from population averages. This “one drug fits all” approach could, with the fruits of pharmacogenomic research, evolve into an individualized approach to therapy where optimally effective drugs are matched to a patient’s unique genetic profile. This involves classifying patients with the same phenotypic disease profile into smaller subpopulations, defined by genetic variations associated with disease, drug response, or both. The assumption underlying this approach is that drug therapy in genetically defined subpopulations can be more efficacious and less toxic than in a broad population. Individualizing drug therapy raises a number of issues with enormous practical consequences. Currently, the pharmaceutical industry is in a consolidation and merger phase, with ever larger corporations emerging at a steady pace. This consolidation is done in the expectation that many novel drugs can be brought to market with high efficacy against major diseases, driven by genomics-based drug discovery. Indeed, large corporations depend on generating “blockbuster” drugs-; drugs that raise in excess of a billion dollars in revenue each year by targeting large patient populations. However, it remains to be seen whether betting on a “one drug fits all” approach is realistic. Certainly, a few blockbuster drugs continue to emerge, for example, the Cox-2 selective inhibitors in the therapy of inflammatory joint diseases. Efficacy does not appear to exceed substantially that of traditional nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit both Cox-1 and Cox-2 to varying degrees; however, the incidence of gastrointestinal lesions is reduced. Yet, only a portion of patients receiving conventional NSAIDs develop these lesions, and the traditional drugs are much less expensive. Moreover, it remains to be seen what long-term sequelae arise from treatment with Cox-2 selective inhibitors. These sequelae might be beneficial (for example, the possible prevention of colon cancer or neurodegenerative disorders associated with inflammation in the CNS), but the physiological functions of Cox-2 remain poorly understood. Trials over longer time periods will be needed to address these questions fully. As three quarters of all health care costs are used for the treatment of chronic illness, mostly of the aged, long-term issues will be the battleground where optimal therapies will be decided. Whether a single drug emerges superior to others in a broad patient population or whether best clinical response requires differential therapy of small subpopulations is the subject of fierce debates. Bringing a new drug to the market currently costs approximately $500 million, making it economically impossible to target small patient populations. If smaller patient populations are to be served, we need to change the entire process, up to final regulatory agency approval for clinical use. Conceivably, targeting well-defined patient populations will sharpen our analysis of risk/benefit ratios and permit clinical trials to be substantially reduced in size. Laws and FDA regulations may have to be changed to accommodate the need for

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Personalised Medicine and Pharmacogenomics

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targeting patients with rare diseases or with subtypes of otherwise common diseases. This approach will set the stage for testing whether targeting small patient populations with select drugs is superior to treating many patients with the best drug available for a given disease. The outcome may vary from one case to another. Thus, individualizing drug therapy with the use of pharmacogenomics holds the potential to revolutionize medical therapeutics, by challenging the “one drug fits all” approach. Furthermore, pharmacogenomics could also enhance the value of currently approved drugs with limited market share because of significant toxicity or limited efficacy, enabling prescribers to identify patients for whom they will be both effective and safe. The emergence of personalized medicine raises issues for those who pay for treatment. The cost of new diagnostic tests and individualized medications may be more expensive, but it is hoped that the predictive potential of personalized medicine could avert more costly treatments required after the onset of a disease. Currently, less than 5% of all US private companies reimburse for genetic tests, indicating that the current health care delivery system may not be able to deliver effective “personalized medicine”. Pharmacogenetics applies not only to traditional drugs but also to bioengineered proteins and gene therapy. Human genetic variability can be expected to affect all treatment modalities. For example, breast cancer treatment with trastuzumab (Herceptin), a humanized monoclonal antibody against the HER2 receptor developed by Genentech, Inc., is linked to HER2 overexpression. This reaction correlates with poor clinical prognosis and serves as a marker for responsiveness to trastuzumab therapy, either alone or in combination with chemotherapy. The cytochrome P450 monooxygenase system of enzymes is responsible for a major portion of drug metabolism in humans. Although commonly serving to detoxify xenobiotics, these enzymes are also principally responsible for the activation of procarcinogens and promutagens in the human body. This scenario is particularly important for lipophilic drugs such as CNS-active drugs, which generally must be lipophilic to penetrate the blood-brain barrier. Because renal excretion is minimal for these compounds, P450 metabolism provides the primary means of drug elimination. This large family of genes has been intensely studied, and among the numerous P450 subtypes, CYP2D6, 3A4/3A5, 1A2, 2E1, 2C9, and 2C19 play particularly critical roles in genetically determined responses to a broad spectrum of drugs. Determination of a patient’s CYP2D6 phenotype/genotype may prove useful in treatment with antipsychotic drugs, while comprehensive genotyping assays for all relevant P450 isotypes and their main sequence variants are being developed. Cytochrome P450s inactivate or in some cases activate xenobiotics. Therefore, P450 polymorphisms affect an individual’s susceptibility to environmental toxins. As a result, sequence variation of P450 isotypes attracts special attention in toxicogenetics. Recently the US National Institute of Environmental Health Sciences launched the Environmental Genome Project with the stated goal of understanding the genetic factors governing an individual’s response to the environment on a genome-wide scale. This effort parallels the study of genetic variability in drug response.

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Examples of personalized cancer management include: • Testing for disease-causing mutations in the BRCA1 and BRCA2 genes, which are implicated in familial breast and ovarian cancer syndromes. Discovery of a disease-causing mutation in a family can inform “at-risk” individuals as to whether they are at higher risk for cancer and may prompt individualized prophylactic therapy including mastectomy and removal of the ovaries. This testing involves complicated personal decisions and is undertaken in the context of detailed genetic counseling. • Minimal residual disease (MRD) tests are used to quantify residual cancer, enabling detection of tumor markers before physical signs and symptoms return. This assists physicians in making clinical decisions sooner than previously possible. • Targeted therapy is the use of medications designed to target aberrant molecular pathways in a subset of patients with a given cancer type. For example, Herceptin is used in the treatment of women with breast cancer in which HER2 is overexpressed. Tyrosine kinase inhibitors such as Gleevec have been developed to treat chronic myeloid leukemia (CML), in which the BCR-ABL fusion gene (the product of a reciprocal translocation between chromosome 9 and chromosome 22) is present in >95% of cases. These medications specifically inhibit the Ableson tyrosine kinase (ABL) and are thus a prime example of “rational drug design” based on knowledge of disease pathophysiology.

23.9 Personalized Medicine and Diseases Personalized medicine aims to identify individuals at risk for common diseases such as cancer, heart disease, and diabetes. The simple family history has long been used by physicians to identify individuals at increased risk and to advise preventive measures such as lifestyle modifications (changes in diet, cessation of toxic habits, increased exercise) earlier screening, or even prophylactic medications or surgery. Scientific advancements offer the potential to define an individual’s risk based on their genetic make-up. Fields of biomedical research termed “-omics” (genomics, proteomics, and metabolomics) study the contribution of genes, proteins, and metabolic pathways to human physiology and variations of these pathways that can lead to disease susceptibility. It is hoped that these fields will enable new approaches to diagnosis, drug development, and individualized therapy. Nanotechnology will also play a major role in this area (Jain, 2005).

23.10 Personalized Medicine and Diagnostics Industry Although pharmacogenomics-based diagnostics and therapeutics are increasingly being translated into personalized medicine applications, relatively little evidence exists about how novel pharmacogenomics-based technologies will be accepted and

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Personalized Medicine and Diagnostics Industry

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adopted by patients. It is important to understand the characteristics of genomic diagnostics and targeted therapeutics that might impact utilization or serve as barriers to adoption of these novel technologies in order to formulate appropriate policies and procedures. The traditional diagnostics industry is mature and only achieving a high growth rate. The diagnostics industry has not been as successful as the pharmaceutical industry in attracting investment funding. However, the advent of molecular diagnostic tests, or theranostics, opens new opportunities in a small but believed to be rapidly growing niche market. New relationships are likely to develop between industry partners committed to personalized medicine embracing the approach of successful, specialised pharmaceutical firms. Major pharmaceutical firms have responded to the growing emphasis on individualized therapy to improve drug efficacy and safety with large investments in pharmacogenomics research ( Posner, 2005). It is becoming apparent that genetic testing to identify patients in whom a particular drug can be given safely and effectively may provide those products with a competitive advantage. Several of the world’s largest pharmaceutical firms, including AstraZeneca, Bayer, Pfizer, SmithKline Beecham, and Novartis have formed a consortium with 5 major academic centers with the goal of identifying 300,000 heritable SNPs within the next 2 years. The National Institutes of Health, in an independent effort, has made $30 million available over 3 years, starting in January of 1998, for the discovery and compilation of 100,000 SNPs. To top this all, scientists at Celera Genomics contend that they will have a collection of 6 to 10 million SNPs by mid 2000. With availability of high-resolution SNP maps and DNA microarray analytical capability, performing genome-wide association studies during clinical trials becomes feasible, enabling one to identify disease-susceptibility genes for prognosis, drug discovery, and selection of therapy. If risk for a given disease is predicted to be high, as judged by the SNP pattern of a patient, preventive therapy and lifestyle adjustments (diet, exercise, etc) may be implemented. A comprehensive SNP map will also contain genetic variants relevant to drug transport, metabolism, and receptor interaction and, therefore, needs to be considered in drug selection. Moreover, a comprehensive SNP map may also serve to alert the therapist when careful drug dosage monitoring is required. Stratifying patient populations using genome-wide SNP maps presents a major challenge to the pharmaceutical industry. The outcome from applying such an approach cannot be accurately gauged at present. Today, many examples of variability in both drug response and toxicity associated with known genetic variability are documented. In a few cases, genetic tests are beginning to find their way into clinical practice, making a proactive approach to individualized therapy possible. In cancer chemotherapy of acute lymphocytic leukemia, administration of drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine can cause severe hematologic toxicity or even death in patients possessing nonfunctional (“null”) variants of thiopurine methyltransferase (TPMT). Functional assays of TPMT in red blood cells, or alternatively genotyping, can identify those patients (approximately 1 in 300) who are homozygous for alleles encoding nonfunctional enzyme, and therefore unable to metabolize the drugs to

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their inactive methylated forms. These patients can be safely treated with doses 10–15 times less than commonly prescribed. Therefore, genotyping, or functional enzyme analysis, has become standard practice in major cancer treatment centers such as the Mayo Clinic (Rochester, MN) and St. Jude’s Children Research Hospital (Memphis, TN). Nanobiotechnology is being used to refine discovery of biomarkers, molecular diagnostics, drug discovery and drug delivery, which are important basic components of personalized medicine and are applicable to management of cancer as well. Examples are given of the application of quantum dots, gold nanoparticles, and molecular imaging in diagnostics and combination with therapeutics another important feature of personalized medicine. Personalized medicine is beginning to be recognized and is expected to become a part of medical practice within the next decade. Personalized management of cancer, facilitated by nanobiotechnology, is expected to enable early detection of cancer, more effective and less toxic treatment increasing the chances of cure (Jain, 2005).

23.11 Impact of the US Patent System on the Promise of Personalized Medicine The pharmaceutical industry faces a number of challenges that have cumulatively led to a decline in productivity, despite increasing levels of investment in research and development (Posner, 2005). The US patent system rewards innovation in medicine and other arts and sciences by granting innovators, for a period of time, the right to exclude others from using what was invented. One of the purposes of the patent system is to trade that right to exclude, and in its stead obtain the patent holder’s obligation to fully and publicly disclose the essence of the innovations so that they can be improved, thus advancing the common welfare. A tension exists between personalized medicine’s need for access to and use of scientific advances and the patent system’s reward of exclusive use or nonuser to innovators. This tension may result in fewer diagnostic and therapeutic tools brought to the market and generally adopted. The risk seems particularly acute with respect to the diagnostic and therapeutic tools arising from genetic testing that hold specific value for a subset of the population. The judicial system has introduced ethical exceptions that overcome a patent holder’s right to exclude; these judicial overrides relate to the provision of certain types of medical procedures and the development of certain types of new drugs, and not, apparently, to the use of diagnostic and therapeutic tools essential to the success of personalized medicine. A serious question exists as to whether legislative action is necessary to increase public access to genetic testing (Solomon and Sieczkiewicz, 2007). Personalized medicine is a paradigm that exists more in conceptual terms than in reality, with only a few marketed drug-test companion products and not very many actual clinical practices set up to personalize medicine in the way that supporters have intended. Nevertheless, the reality of personalized medicine has become more imminent because of the increased awareness of the shortcomings in the delivery of drugs with adequate benefit/risk to patients, a better molecular understanding of

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Ethical Legal and Social Issues of Personalised Medicine

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how to optimize drug selection and dosing, and an increased demand for integrating more clinically relevant genetic information into the drug development process to improve both innovation and productivity,. “personalized medicine” is in many ways simply an extension of traditional clinical medicine taking advantage of the cutting edge of genetics research. However, obstacles to the implementation of this vision are formidable. The dynamic complexity of the human genome, multigenic disease origins, and involvement of numerous genes in drug response impede the effective application of genome-wide SNP scanning in the clinic. Drug responses will most likely be associated with patterns of multiple polymorphically expressed traits, rather than single causative polymorphisms. Such patterns of genetic variants differ among distinct ethnic groups. This factor could obscure prediction of disease susceptibility and drug response across patient populations, and it points to the need to genetically stratify patients for clinical pharmacogenomic studies.

23.12 Ethical Legal and Social Issues of Personalised Medicine We are uncertain as to the overall direction of pharmacogenomics and personalized medicine over the next 10 years. Although new analytical systems introduced during the last decade have offered incremental improvements over previously available technology, they have not allowed scientists to maximize the benefit of multiple advancements in genomics, combinatorial chemistry, and assay technologies. The realization of an individualized approach to drug discovery and therapy will require new statistical methods and analytical systems providing an order-of-magnitude increase in throughput, along with corresponding decreases in operating costs, with enhanced accuracy and reduced complexity. In addition to the daunting scientific challenges we have outlined, ethical issues need to be resolved. Information about an individual’s genetic makeup raises privacy questions and ethical dilemmas about disease susceptibility, prognosis, and treatment options. Obviously, information of this type must be carefully safeguarded to ensure privacy. Many legal and economic issues will need to be resolved (Jeffords and Daschle, 2001). Whether or not these new genomic technologies find their way into everyday clinical use during the next 10 years, they will prove valuable tools in clinical research directed at optimizing drug therapy. The vision of pharmacogenomics is leading us to a more individualized approach to drug therapy, while revealing limits inherent to the treatment of disease in broad patient populations. Ethical issues are involved in the development of personalized medicine mainly in the area of genetic testing. These along with social issues and consideration of race in the development of personalized medicine are being debated. Regulatory issues are discussed mainly with reference to the FDA guidelines on pharmacogenomics. Increase in efficacy and safety of treatment by individualizing it has benefits in financial terms. Information is presented to show that personalized medicine will be cost-effective in healthcare systems. For the pharmaceutical companies, segmentation of the market may not leave room for conventional blockbusters but smaller and exclusive markets for personalized medicines would be profitable. Marketing

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opportunities for such a system are described with market estimates from 2007 to 2017.

23.13 Conclusion Personalized medicine promises to offer the right treatment for the right patient at the right time. Although that promise might seem far off, there is clear evidence that the traditional trial-and-error practice of medicine is eroding in favor of more precise marker-assisted diagnosis and treatment. For the patient, the benefits are clear: safer and more effective treatment of disease. For industry there appears an equally desirable outcome of this approach: increased efficiency, productivity and better product lines. Society as a whole will also realize a benefit: more focused application of precious healthcare resources to those in need of them most. The realization of personalized medicine is not without challenges, yet many of these challenges are being addressed. By encouraging public dialogue and debate, we expect that there will be continued progress forward. Lastly, as we take on more and more of the burden of our own health and well-being, educational forums must be developed for patients and physicians alike to understand the complex nature of the genomic information that is being used for decision making. Then we will have truly fulfilled the promise of the future.

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classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286:531–537. Halushka, M.K., Fan, J.B., Bentley, K., Hsie, L., Shen, N., Weder, A., Cooper, R., Lipshutz, R., Chakravarti, A., 1999, Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet 22:239–247. Hedenfalk, I., Duggan, D., Chen, Y., Radmacher, M., Bittner, M., Simon, R., Meltzer, P., Gusterson, B., Esteller, M., Kallioniemi, O.P., Wilfond, B., Borg, A., Trent, J., Raffeld, M., Yakhini, Z., Ben-Dor, A., Dougherty, E., Kononen, J., Bubendorf, L., Fehrle, W., Pittaluga, S., Gruvberger, S., Loman, N., Johannsson, O., Olsson, H., Sauter, G., 2001, Gene-expression profiles in hereditary breast cancer. New Engl J Med 344:539–544. Issa, A.M., Tufail, W., Hutchinson, J., Tenorio, J., Baliga, M.P., 2009, Assessing patient readiness for the clinical adoption of personalized medicine. Public Health Genomics 12: 163–169. Jain, K.K., 2002, Personalized medicine. Curr Opin Mol Ther 4:548–558. Jain, K.K., 2005, Role of nanobiotechnology in developing personalized medicine for cancer. Technol Cancer Res Treat 4:645–650. Jeffords, J.M., Daschle, T., 2001, Political issues in the genome era. Science 291:1249–1251. Kohane, I.S., 2009, The twin questions of personalized medicine: who are you and whom do you most resemble?. Genome Med 1:4. Lazarou, J., Pomeranz, B.H., Corey, P.N., 1998, Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. J Am Med Assoc 279:1200–1205. Nohmi, T., 2002, Drug discovery based on genomic information. Rinsho Byori 50:694–697. Page, M.J., Amess, B., Townsend, R.R., Parekh, R., Herath, A., Brusten, L., Zvelebil, M.J., Stein, R.C., Waterfield, M.D., Davies, S.C., O‘Hare, M.J., 1999, Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties. Proc Natl Acad Sci USA 96:12589–12594. Posner, B.A., 2005, High-throughput screening-driven lead discovery: meeting the challenges of finding new therapeutics. Curr Opin Drug Discov Devel 8:487–494. Priori, S.G., Barhanin, J., Hauer, R.N., Haverkamp, W., Jongsma, H.J., Kleber, A.G., McKenna, W.J., Roden, D.M., Rudy, Y., Schwartz, K., Schwartz, P.J., Towbin, J.A., Wilde, A.M., 1999, Genetic and molecular basis of cardiac arrhythmias: Impact on clinical management. Part III. Circulation 99:674–681. Richmond, T.D., 2008, The current status and future potential of personalized diagnostics: Streamlining a customized process. Biotechnol Annu Rev 14:411–422. Sadee, W., 2002, Pharmacogenomics: the implementation phase. AAPS PharmSci 4:E5. Schwardt, O., Kolb, H., Ernst, B., 2003, Drug discovery today. Curr Top Med Chem 3:1–9. Schwartz, P.J., Priori, S.G., Locati, E.H., Napolitano, C., Cantù, F., Towbin, J.A., Keating, M.T., Hammoude, H., Brown, A.M., Chen, L.S., 1995, Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na? Channel blockade and to increases in heart rate: implications for gene-specific therapy. Circulation 92:3381–3386. Shimizu, W., Antzelevitch, C., 1997, Sodium channel block with mexiletine is effective in reducing dispersion of repolarization and preventing torsade de pointes in LQT2 as well as LQT3 models of the long-QT syndrome. Circulation 96:2038–2047. Solomon, L.M., Sieczkiewicz, G.J., 2007, Impact of the US Patent System on the promise of personalized medicine. Gend Med 4:187–192. Triggle, D.J., 2007, Drug discovery and delivery in the 21st century. Med Princ Pract 16:1–14. Tsongalis, G.J., Cartun, R.W., Ricci, A. Jr., 2000, Gene amplification as means for determining therapeutic strategies in human cancers. Clin Chem Lab Med 38:837–839. Van Eldik, L.J., Koppal, T., Watterson, D.M., 2002, Barriers to Alzheimer disease drug discovery and development in academia. Alzheimer Dis Assoc Disord 16(Suppl 1):S18–S28. Veer, L.J., Bernards, R., 2008, Enabling personalized cancer medicine through analylsis of geneexpression patterns. Nature 452(7187): 564–570.

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Chapter 24

Biopharmaceutical Industry and Health Care

Abstract Global manufacturing of biopharmaceuticals has increased significantly over the last decade due to a number of reasons. Biopharmaceuticals offer several advantages such as highly effective and potent action, fewer side effects and the potential to actually cure diseases rather than merely treat the symptoms. These advantages, combined with the increasing number of new diseases that can be treated with biopharmaceuticals, are driving enhanced production of these drugs worldwide. Keywords Biopharmaceuticals · Health care · Recombinant protein · Industry · Molecular farming · Pharmaceutical industry · Mimetics and peptidomimetics · Glycotherapeutics · Human monoclonal antibodies

24.1 Prologue Today, the pharmaceutical industry is experiencing a profound transition. Globalization and technological advancement represent the principal pressures for change in the market, where it is increasingly more difficult for this type of industry to efficiently recoup the growing cost of innovation (Gonzalez Pier, 2008). The pharmaceutical industry maintain its dominant position in small molecules through the use of biotechnology in drug discovery, but the development and manufacture of protein based drugs requires a completely different set of core competencies. Broadly, the history of pharmaceutical biotechnology includes Alexander Fleming’s discovery of penicillin in a common mold, in 1928, and the subsequent development, prompted by World War II injuries of large-scale manufacturing methods to grow the organism in tanks of broth. Pharmaceutical biotechnology has since changed enormously. Two breakthroughs of the late 1970s became the basis of the modern biotech industry: the interspecies transplantation of genetic material, and the fusion of tumor cells and certain leukocytes. The cells resulting from such fusion, hybridomas replicate endlessly and can be geared to produce specific antibodies in bulk. Modern pharmaceutical biotechnology encompasses gene cloning and recombinant DNA technology. Gene cloning comprises isolating a segment of DNA M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_24,

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molecule that corresponds to a single gene and synthesizing (“copying”) the segment (Steinberg and Raso, 1998).The first drug produced via genetic engineering was human insulin which appeared on the market in 1982. By mid-2000, 84 biopharmaceuticals had been approved for marketing with almost half launched during the past three years. Worldwide sales have grown more than seven-fold over the past decade to reach US$15 billion by 1998. The US represents 46% of the market, compared to 36% for conventional drugs, due to a combination of earlier regulatory approval, easier market acceptance, and greater pricing flexibility than other countries. Although biopharmaceuticals comprise only 5% of world prescription drug sales, they account for six of the top 50 selling drugs, 13% of new medicines approved by the FDA in the 1990s and about 18% of all drugs in development. At the end of 1999, there were 369 biotechnology drugs in US clinical development against 438 disease indications with 25% in Phase III. Pharmaceutical policy offers a rare example for a complementary approach between a sound health policy and an efficient economic policy; that is, a “healthy pharmaceutical policy.”

24.2 Concept The discovery of recombinant DNA and monoclonal antibody technologies in the 1970s marked the birth of the biopharmaceutical industry. Biopharmaceuticals are complex macromolecules derived from recombinant DNA technology, cell fusion, or processes involving genetic manipulation. They include recombinant proteins, genetically engineered vaccines; therapeutic monoclonal antibodies; and nucleic acid based therapeutics (i.e. DNA based drugs), including gene therapy vectors. Unlike orally delivered small molecule drugs that underpin the traditional pharmaceutical industry, biopharmaceuticals are usually administered by subcutaneous, intravenous, or intramuscular injection (Hu et al., 2006). Biopharmaceutical is a subset of pharmaceutical. Biopharmaceuticals are pharmaceuticals inherently biological in nature and manufactured using biotechnology (involving use of live organisms), while drugs comprise the other major subset of pharmaceuticals, with their source and manufacture being chemical (non-biological) in nature. These molecules manufactured by biotechnology methods, with the products obviously having biological sources, usually involving live organisms or their active components (bioprocessing; also usually very obvious; or directly involving surrogates, e.g., protein/gene sequences). This broad view has been adopted by Biopharmaceuticals in the U.S. and European Markets, which includes all recombinant proteins, (monoclonal) antibodies, vaccines, blood/plasma-derived products, nonrecombinant culture-derived proteins, and cultured cells and tissues. Diagnostics, vaccines and recombinant therapeutic proteins are the three main segments in bio pharma industry. Since the last two decades emphasis in new drug development has shifted from small-molecule chemicals to large-molecule proteins and other biopharmaceuticals such as human insulin, gene therapies and specialized antibiotic treatments. Unlike

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Biopharmaceutical Research

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chemically synthesized small molecule drugs that have long underpinned the traditional pharmaceutical industry, biopharmaceuticals are complex macromolecules created through the genetic manipulation of living organisms using gene cloning, recombinant DNA (gene splicing), or cell fusion technologies. In terms of product type, these include: recombinant proteins; recombinant antigen vaccines and vaccines crafted from genetic material such as DNA; therapeutic monoclonal antibodies; and oligonucleotides (short sequences of DNA or RNA) such as antisense molecules which interrupt the production of disease causing proteins by inhibiting gene function and gene therapy which can enhance the production of a missing protein through the addition of a synthetic gene. The first recombinant protein, human insulin was launched in 1982, the first recombinant vaccine against hepatitis B in 1986, the first therapeutic monoclonal antibody against kidney transplant rejection also in 1986, and the first and only oligonucleotide in 1998 (against cytomegalovirus retinitis in AIDS patients). No gene therapy product has yet been approved. Recombinant proteins dominate the biopharmaceutical market accounting for the bulk of sales to date. The biopharmaceutical industry cannot be identified using the Standard Industrial Classification which distinguishes firms on the basis of their output rather than their technology or production process, and international data are difficult to compare. Many biotechnology firms, for example, are identified as biopharmaceutical companies to differentiate them from the mainstream pharmaceutical industry, but their prime focus are small molecule drugs targeted against proteins thought to be important in the disease pathway (proteins can be used both as drugs i.e. biopharmaceuticals or drug targets). Biologics, an area that consists of blood derived polyclonal antibodies and clotting factors, antibiotics, and classical vaccines based on live or killed viruses are frequently classified as biopharmaceuticals, but these long predate the emergence of recombinant DNA and monoclonal antibodies. Insulin, for example, was originally obtained from porcine or bovine pancreas while human growth hormone was extracted from the pituitary glands of cadavers. The biopharmaceutical category also often includes drugs derived from plants, fungi or marine organisms, but these are more in the realm of traditional medicinal chemistry research based on the random screening of natural compounds.

24.3 Biopharmaceutical Research Biopharmaceutical research encompasses a wide range of scientific disciplines: genetics, molecular biology, biochemistry, microbiology, physics, pharmacology, and information technologies. Modern drug discovery is built on four core technologies: genomics (source of novel targets), combinatorial chemistry (source of molecules that interact against those targets), high-throughput screening (testing one against the other); and bioinformatics which is crucial to the analysis of the vast amounts of data generated. While current pharmaceuticals are active against some 500 biological targets, genomics is expected to lead to the identification of up to 10,000 new targets for development (Dutta and Garner, 2003). Whether these

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will be amenable to small molecule intervention or require protein and nucleic acid based therapies remains to be seen. Despite the heavy investment in genomics, only a handful of genomics-based drugs (defined as those based on the identification of an unknown gene sequence followed by elucidation of its function and therapeutic potential) have reached the clinical stage. All are protein drugs.

24.4 Opportunities in Healthcare The biopharmaceuticals industry is a rapidly growing sector within the pharmaceuticals industry with immense opportunities and implications for healthcare. The success achieved by companies with a few revolutionary new drugs has brought this industry into the limelight in recent years. However, the demand and supply equation for manufacturing capacities has recently become a cause for much speculation. The capacity gap, the difference between the demand and supply of manufacturing capacities represents a fundamental challenge to the industry (Serajuddin and Serajuddin, 2003). Deficit of capacity is expected to significantly impede industry progression. Quantifying and understanding this capacity gap is essential for biopharmaceuticals developers. Developers also need to examine and evaluate their own financial positions. This will enable them to decide whether to outsource the manufacturing to contract manufacturing organisations chiefly on long-term basis or invest in setting up manufacturing plants. Manufacturing capacity supply is expected to be greater than the worldwide demand throughout 2005–2011. The global manufacturing capacity of biopharmaceuticals was around 2.27 million litres in 2004. This includes the capacity held by both captive use and contract manufacturers. It is expected to increase to 3.69 million litres in 2011 at a compound annual growth rate (CAGR) of 7.2%. The annual biopharmaceutical technology infrastructure expenditures are shown in Fig. 24.1. Today indeed, the discovery and marketing of innovative medicines is linked: not only to Companies abilities to solve relatively basic and traditional difficulties which have deeply changed over the past few years, for instance: the regulatory constraints, the dramatic increase in development costs, the critical mass required for investments, the development time periods and the protection of innovation, human resources and organization; but also to the acceptance of recent factors which the Research Pharmaceutical Industry must imperatively overcome, such as: the new Research channels and namely biotechnologies, the building of a pharmaceutical Europe and the internationalization of molecules, the needs of the third world, the innovation financing and the control of Health expenditures, the public opinion’s shift in expectations regarding ethical and environmental issues. The Pharmaceutical Industry is currently in a shake-up phase during which it will undergo deep changes, with regard both to its activities and in relations with its environment (Chalchat, 1993). The Research Pharmaceutical Industry is currently facing a number of increasingly complex challenges.

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Molecular Diagnostics and Health Care

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Fig. 24.1 Annual biopharmaceutical technology infrastructure expenditures

The high cost of biopharmaceuticals and the low reimbursement levels from insurance companies present a major challenge. Companies need to explore various strategies to counter this challenge such as developing technologies that bring down the cost of drugs as well as manufacturing pharmaceuticals with substantial market size.

24.5 Molecular Diagnostics and Health Care Since the last century pharmaceuticals has been an industry of intensive innovation. (Marini, 2006). The rate and direction of technological innovation are affected by a complex of supply- and demand side factors, including biomedical research, education, patent law, regulation, health care payment, law, and more (Goodman and Gelijns, 1996). Molecular biology has revolutionized the understanding of many aspects of human disease. Ongoing developments in DNA diagnostics, the analysis of disease at the nucleic acid level will soon provide automated, rapid, and inexpensive analyses for DNA or RNA sequences associated with genetic, malignant, and infectious diseases. DNA diagnostics will also facilitate the identification of disease-associated genes at birth, thus creating new opportunities for preventive medicine. Pharmaceutical products can be categorized according to their intended purpose that is, diagnostic (to aid in the detection of a disease), therapeutic (treatment), and vaccines and other biological products (prevention). Products included in the diagnostics category are instruments and reagents used for the screening, diagnosis, and monitoring of diseases. They consist, for the most part, of immunoassays

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(tests for hormones, allergies, HIV, etc.), clinical chemistry (enzymatic, electrochemical, and chromatographic techniques), hematology testing (blood counts), diabetes (omnipresent on the home-testing market), and microbiology tests (supplies of bacterial cultures and various probe tests for specific microorganisms). From the industry’s standpoint, therapeutic drugs are typically labelled according to their patent status. For instance, patented drugs (also known as innovative or brand-name drugs) provide a manufacturer with the exclusive right to make and sell a drug for a certain period of time. Nonpatented drugs, in contrast, refer to generic copies of existing patented drugs and other specialty products that were previously subject to patent protection (Grabowski, 2004). But therapeutics also includes natural health remedies, the functional foods and nutraceuticals technology. Their emergence into the sphere of pharmaceuticals can be attributed, among other things, to the increasing use of traditional medicines derived from plants, herbs, and other natural sources as alternatives to modern medicine and pharmaceuticals. Finally, the emergence of biotechnologies has also injected new life into vaccines and other biological products. Combined with the fact that vaccination is no longer limited to infants, a growing number of companies are focusing their resources on the development of new and improved vaccines. Advances in vaccines against sexually transmitted diseases, flu vaccines, adjuvant (i.e., substances pooled with antigens to enhance the immune response), and hightech vaccines such as naked DNA vaccines (which involve genes instead of proteins) are expanding the boundaries of this field. In addition to vaccines, this group of products contains other biological materials such as plasma and blood products, insulin (a hormone produced by the beta cells of the pancreas that helps to regulate the amount of glucose in the blood), as well as scores of other hormones, serums, and enzymes.

24.6 Global Context Already, bioengineered drugs against hairy cell leukaemia as well as medicines for heart attacks (thrombolytic agents), Crohn’s disease, and rheumatoid arthritis (TNF α antibody) and numerous vaccines against infectious agents (hepatitis B virus, HIV, etc.) have been approved. So far, approximately sixty-three biotechnology products have been approved and are available on the market. More than ever, the influx of biotechnologies is breathing new life into the pharmaceutical industry, increasing the efficiency of drug development, and suggesting innovative avenues of research. This is a very welcome sign for companies that are increasingly feeling the pressures of bottom-line financial performances and rising cost containment measures. The World’s Top Fifteen Pharmaceutical Companies includes United States Merck & Co., Bristol-Myers Squibb, Pfizer, American Home Products, Johnson & Johnson, Lilly, Abbott, Schering-Plough, United Kingdom Glaxo Wellcome, AstraZeneca, SmithKline Beecham, Switzerland Novartis, Roche, Germany Aventis, France Sanofi-Synthélabo.

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Global Context

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Products and markets: By mid-2000, 84 biopharmaceuticals had been approved for marketing with almost half launched during the past three years. Worldwide sales in last 10 years ago totalled US$15 billion now it is much more. Although representing only 5% of world prescription drug sales of $300billion, biopharmaceuticals comprise six of the top 50 selling drugs, 13% of new medicines approved by the FDA in the 1990s, and 11% of all drugs in development. The US is the largest and most rapidly growing market, accounting for 46% of sales, compared to 36% for conventional prescription drugs, reflecting a combination of earlier regulatory approval, easier market acceptance and greater pricing flexibility. Europe and Japan are the next most important markets, accounting respectively for 30 and 17% (Federsel, 2006). Industry size: There are approximately 3000 dedicated biotechnology companies worldwide employing 230,000 people with the US accounting for 40% of firms and 70% of employment. Publicly traded firms – including an estimated 100 that floated IPOs during 2000 – number approximately 460 of which 74% are based in the US and 18% in the EU. The segment involved in biopharmaceuticals numbers about 150 firms. Only a handful of biotech firms – primarily those involved in biopharmaceuticals- have a product on the market. The industry as a whole continues to experience major losses because of high R&D costs. The largest and most successful (e.g. Amgen, Chiron, Biogen and Genentech) are mainly US based. With the exceptions of Serono (Switzerland), Celltech (UK), and Bio-Technology General (Israel), few non-US firms have brought a biopharmaceutical to market, reflecting their later startup dates. The total market capitalization of all public European biotechnology companies, for example, is only a little larger than Amgen. Japan has few dedicated biotechnology companies, with the industry primarily consisting of large food and pharma firms with historic strengths in fermentation attempting to diversify into biopharmaceuticals. There has been a steady migration of European R&D to the US because the US is the most profitable pharmaceutical market in the world, has a more rapid uptake of new drugs, and pricing is largely unregulated by the federal government. As the major European pharmaceutical companies have research operations in the US and are therefore closely tuned to US technical developments, they have looked first to the US biotech industry for collaborative venture, making it difficult to attract investment to Canada. However, factors such as Canadian research excellence, lower wages and infrastructure costs, and access to capital for Québec based start-ups proved instrumental in attracting companies such as Intellivax, Methylgene, DSM Biologics, and Amgen. A number of them have also executed major alliance deals attesting to the commercial of their technology: SemBioSys/Dow agro Sciences (US$12 million) for molecular farming; GlycoDesign/Seikagaku (US$56 million) for anti-inflammatory agents; Inflazyme/Aventis Pharma (US$91 million) for respiratory diseases; and Xenon Genetics/Warner Lambert (US$58 million) for elevating high-density lipoprotein to clear cholesterol from blood. 369 biotechnology drugs were in US clinical development against 438 indications at the end of 1999, with one quarter in Phase III. The three largest categories were vaccines (27%), monoclonal antibodies (16%), and gene therapy (7%). Cancer, mainly breast,

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melanoma, and prostate, accounted for 40% of indications. Canadian products in the survey included human parathyroid hormone for osteoporosis (NPS Allelix); GMCSF – granulocyte-macrophage colony-stimulating factor (Cangene); stress protein against papillomaviris-related dysplasia (StressGen); group A Strep vaccine (ID Biomedical); breast cancer vaccine (Biomira); and anticancer oligonucleotides (Inex). Most biotechnology firms do not have the interest or resources in bringing a product to market, preferring instead to focus on research and early clinical development where they feel they can add the most value; commercialization for these companies means developing the technology to a point where it can be licensed out using royalties to fund the next generation of products. Manufacturing: Only a handful of companies (e.g. Amgen, Genentech, Biogen) have in-house manufacturing capability. The majority contract out their production needs because it is faster and less risky than building a facility and ramping up expertise in-house. Proteins are more difficult to manufacture than conventional drugs, and the time and cost of building and validating a facility certified to current Good Manufacturing Practices (cGMP) standards (up to three years and US$100 million) is a costly and risky commitment, particularly if there is only a single product in advanced stages of clinical development. An added challenge for Canadian companies is the scarcity of bio-chemical engineers with expertise in taking products from the bench through to pilot and commercial scale. The contract biomanufacturing industry is facing capacity constraints due to the large number of protein-based drugs in development combined with the market success of monoclonal antibodies and their great demands for mammalian cell culture. Biotech firms are beginning to queue up for production time and long lead times are expected over the next few years, threatening to increase the time and costs of product development. A number of factors will affect future capacity: the success rates of the products presently in the clinic, actual dose levels of these drugs, the possibility of improving existing manufacturing yields, facility expansions, and the success of molecular farming technologies.

24.7 Emerging Biopharmaceuticals The success of the pharmaceutical industry will continue to depend on its ability to satisfy the clinical needs of established market economies. The number and quality of new drugs emerging from development pipelines seems likely to rise due to increased research and development budgets of the merged pharmaceutical companies, efficiencies across all facets of the development process, increasing use of new technologies and availability of new targets from the ongoing work on the role of human genes in disease pathways. In addition to the traditional small-molecule drugs, the market for protein products, including monoclonal antibodies and therapeutic vaccines, is likely to expand as advances in recombinant and formulation technologies are made. Current work on relatively newer fields of pharmaceutical research, such as novel G-protein-coupled receptors, chemokines/cytokines,

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Emerging Biopharmaceuticals

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integrins and control of cell cycle regulation and signal transduction pathways (kinases, phosphatases and transcription factors) will lead to new drugs over the next decade. Some of the emerging biopharmaceuticals are 1. Mimetics and peptidomimetics (small molecule compounds that mimic proteins and peptides) have advantages over proteins in terms of ease of delivery and cost, but there are considerable technical challenges in mimicking the large and intricate protein-protein interacting surfaces and overcoming the weak binding forces of small molecules, a major cause of side effects. Examples include mimetics of insulin and granulocyte colony stimulating factor (in early stage development), peptide based mimetics of erythropoietin (late stage), and Montreal based Neurochem’s mimetics of glycosaminoglycans (GAGs – complex carbohydrates that promote amyloid fibril formation characteristic of Alzheimer’s) which compete with naturally occurring GAGs to inhibit deposition of amyloids (early stage). 2. Glycotherapeutics (complex sugars/carbohydrates) have significant potential both as drugs and drug targets because of their diverse biological roles. Cell surface carbohydrates act as binding sites for other molecules, playing structural roles in cancer transformation, immune system regulation, tissue repair, and antiinfection responses, as well as genetic disorders such as Gaucher’s and Fabry’s diseases and their enormous structural diversity (more than 10 million polysaccharides can be formed from nine common monosaccharides compared to 16,000 peptides from 20 amino acids). However, their application as biopharmaceuticals has lagged far behind protein drugs because they are difficult molecules to analyse and synthesize. 3. Fully human monoclonal antibodies First generation mouse-based antibodies provoked a strong immune reaction in humans limiting their clinical utility. Strategies to generate more human compatible antibodies include mouse-human chimeras (60–70% human protein) first introduced in 1995, followed by humanized antibodies (90–95% human protein) in 1998 in which certain mouse antibody amino acid fragments are grafted onto a human antibody. The next generation now in clinical development are fully human antibodies produced via methods such as transgenic mice containing human antibody genes, phage display, or collection of human B cells from infected individuals which are then fused with human tumor cells to create antibody producing hybridomas. Because of their high costs and large dose requirements, advances in manufacturing capability are needed to meet market demand. 4. Vaccines Conventional vaccines based on inactivated (killed) viruses; live, attenuated (weakened) viruses; microbial toxoids; or antigenic fragments (subunit vaccines), have a numberof disadvantages. New vaccine strategies include: • recombinant antigen vaccines where the antigen is produced in bacteria or yeast rather than extracting it from chronic human or animal carriers (e.g. hepatitis B)

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• recombinant vector vaccines using weakened viruses such as poliovirus, vaccinia virus or salmonella bacterium as carriers of the disease causing organism to overcome the inability of inactivated whole antigens or recombinant antigen vaccines to enter cells and express the desired gene product (hepatitis B, HIV, herpes simplex, in development) • DNA vaccines, produced from a core gene of a virus, are effective against those pathogens that change their exterior protein coat by attacking the interior proteins of a virus (HIV, malaria, and a number of anticancer applications are the closest to commercialization); and • RNA vaccines (particularly applicable to yellow fever and polio caused by RNA viruses). Genomics has the potential to improve the effectiveness of existing vaccines and design vaccines for diseases for which no protection currently exists: by sequencing invading bacterial and viral organisms, those critical genes that encode the proteins that cause disease can be identified, leading to the development of novel and more effective antigens. Complete DNA sequences of most human pathogenic viruses and more than 20 bacterial pathogens have already been determined. Vaccines are also being developed as anti-cancer agents, involving attempts to activate immune responses against antigens in the tumour to which the immune system has already been exposed. Research is also being undertaken on edible vaccines e.g. transgenic bananas in order to offer an improved mode of delivery. 5. Gene therapy: Whereas recombinant proteins and antibodies are produced outside the body and administered to the patient, gene therapy has the potential for the body to produce the desired protein on its own if a synthetic gene with the correct DNA sequence for that protein can be inserted into targeted defective cells. A number of major challenges have to be solved: design of a safe and effective gene delivery system, the short duration of gene expression, control over the loci where the transferred gene is expressed, and possible immune reaction caused by the vectors, genes or new proteins. Many efforts now concentrate on conditions that require only transient gene expression, e.g. cancer and coronary artery disease, because sustained gene expression is not necessary once the targeted tumor cells die or new blood vessels grow to bypass blocked arteries. Biopharmaceuticals are typically produced by microbial fermentation (e.g. insulin and human growth hormone in E. coli, recombinant hepatitis B vaccine in yeast) or mammalian cell culture (e.g. tPA, Factor VIII, in Chinese hamster ovary cell lines). Production is not the primary cost driver except for those proteins that must be administered in large amounts e.g. antibodies, or over a period of many years. Transgenic plants, the mammary gland of transgenic animals, and eggs from transgenic chickens may offer more attractive alternatives because of their low cost (factor of 10 to 100 over mammalian cell culture), ease of scale-up, and ability to express complex, large, correctly folded proteins (not possible with microbial fermentation). No products derived via molecular farming have yet been approved. The

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Challenges of the Biopharmaceutical Industry

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most advanced is antithrombin for managing coagulation during open heart surgery expressed in transgenic goats (Phase III completed). Others in clinical development include alpha-1-antitrypsin for cystic fibrosis expressed in sheep (Phase II); alpha glucosidase against Pompe’s disease expressed in transgenic rabbits (Phase II); and the anti-coagulant hirudin in canola, monoclonal antibodies in corn, and interleukin-10 in tobacco leaves (all in Phase I).

24.8 Challenges of the Biopharmaceutical Industry The pharmaceutical industry is investing heavily in new biotechnologies, building this capability internally so it can move from traditional screening and medicinal chemistry capabilities to the front end of drug discovery research, in direct competition with biotechnology firms (Vilian, 1998). Access to capital is critical due to the high costs of drug development and length of time it takes to get a product approved. Most firms do not have a product on the market that can be used to generate cash flow and instead are dependent on funds from venture capital, public equity markets, and strategic alliances used to share development costs and spread risk. The industry predominantly consists of researchoriented, royalty-based companies. Product rights are usually licensed at an early stage with multinational pharmaceutical partner’s gaining most of the commercial benefits. With the exception of recombinant insulin; biopharmaceuticals firms are much more expensive than traditional drugs. There is difficulty developing the data needed on long-term outcomes and evaluating benefits, some of which take the form of quality of life improvements. At the same time, there is a need for governments to recognize the overall health care and social economic benefits in evaluating formulary inclusion. The biotechnology industry has been characterized by significant litigation involving intellectual property rights. Genomic companies have filed for patent protection on thousands of genes including the proteins associated with them which is a major area of concern. Although the US PTO has recently tightened gene patents, it will take court precedents to ultimately clarify the issues. Despite decreases in regulatory approval times in the US and the EU, there has been a substantial increase in the time biopharmaceuticals spend in clinical development, from 39.4 months for products approved in the 1980s to 61.5 months in the late 1990s. A large number have also been discontinued in Phase III. Many of the earlier products were recombinant versions of natural hormones with relatively well understood properties compared to newer biopharmaceuticals with more difficult therapeutic targets and undefined disease mechanisms. Also, the hurdle for discontinuing development is higher for a biotechnology company because it only has a handful of products in clinical development and its stock price is largely determined by its lead product. In short, the pharmaceutical industry is made up of a wide variety of players, each of which engages in one or more functions in the production process (Kola 2008). To understand the industry’s dynamic evolution, however, we need to look at some of the leading trends in pharmaceuticals over the last few years.

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References Chalchat, B., 1993, Current challenges for the pharmaceutical research industry. Ann Pharm Fr 51:16–25. Dutta, A.S., Garner, A., 2003, The pharmaceutical industry and research in 2002 and beyond. Drug News Perspect 16:637–648. Federsel, H.J., 2006, In search of sustainability: process R&D in light of current pharmaceutical industry challenges. Drug Discov Today 11:966–974. Gonzalez Pier, E., 2008, Healthy pharmaceutical policy. Salud Publica Mex 50(Suppl 4): S488–S495. Goodman, C.S., Gelijns, A.C., 1996, The changing environment for technological innovation in health care. Baxter Health Policy Rev 2:267–315. Grabowski, H., 2004, Are the economics of pharmaceutical research and development changing?: productivity, patents and political pressures. Pharmacoeconomics 22:15–24. Hu, X., Ma, Q., Zhang, S., 2006, Biopharmaceuticals in China. Biotechnol J 1:1215–1224. Kola, I., 2008, The state of innovation in drug development. Clin Pharmacol Ther 83:227–230. Marini, G., 2006, Biopharmaceutical industry’s commitment to innovation and to the future of health care. Recenti Prog Med 97:626–633. Serajuddin, H.K., Serajuddin, A.T., 2003, Value of pharmaceuticals: ensuring the future of research and development. J Am Pharm Assoc 46:511–516. Steinberg, F.M., Raso, J., 1998, Biotech pharmaceuticals and biotherapy: an overview. J Pharm Pharm Sci 1:48–59. Vilain, E., 1998, CYPs, SNPs, and molecular diagnosis in the postgenomic era. Clin Chem 44:2403–2404.

Chapter 25

Forensic Medicine

Abstract The knowledge and technique of medical science applied to assist in the resolution of crimes, legal disputes, etc., constitute forensic medicine. Establishing the identity of victims (in cases of murder, accidents, etc.), criminals (in case of rape, murder, etc.) and the father (in case of paternity disputes), etc., is critical in solving the problem of crimes and disputes. This field was completely revolutionized by the technique of DNA fingerprinting. Using this molecular diagnostic technique, the identity of a person can be traced with the help of bloodstain, semen stain, hair roots, tears, saliva and even perspiration. Keywords Forensic medicine · DNA fingerprinting · Pathology · Autopsy · Anthropometry · Pathology · RFLPs · mtDNA · Sex determination · Single locus probe · SLP · STR · Microsatellite · Antemortem · DNA methylation · Clinical forensic medicine · Post mortem

25.1 Prologue In the large majority of homicide trials, there is nothing controversial in the pathologist’s testimony. This is not to say that the testimony is unimportant. It is just that, the medical evidence, usually does not establish the guilt or innocence of the defendant nor does it independently establish a degree of the guilt. What is really important, in the majority of instances, is to provide expert opinion based upon objective, indisputable facts which help to evaluate the reliability and credibility of other witnesses. The pathologist is a middle man between the facts of the crime and the testimony of persons whose version of the episode is at issue. Trust and confidence in scientific evidence have undoubtedly improved over the past few years and forensic sciences themselves have progressed in reliability and effectiveness. Law enforcement agencies rely on scientific investigation to a constantly growing extent. Objectivity, reliability and completeness are the hallmarks of scientific evidence for it to be acceptable in courts. A scientific investigation should have pertinence to the problem under question. Irrelevant scientific investigation will only add to the confusion and may even lead to miscarriage of justice. The quality and reliability of scientific investigations depends on many factors. M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_25,

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Failure to take notice of these factors may result in erroneous conclusions and wrong interpretation of the scientific observations. The investigator, therefore, must take all necessary precautions to see that extraneous factors do not interfere with the scientific experiment and vitiate the findings.

25.2 Concept Forensic medicine and pathology have traditionally been regarded under the auspices of the medical examiner and/or coroner, with medico-legal investigation of unexpected, unexplained, mysterious and/or violent deaths being its principal application. The field of forensic medicine embraces not only traumatic and/or drug-related pathology, however, but also addresses issues of child and elder abuse, sudden and unexpected deaths of a broad constellation, public health and many issues within the overarching fields of the law, medicine and the judiciary. Applications of forensic medicine has impact in almost all fields of clinical medicine to an at least limited degree, with assessment of patient mortality and proper completion of the death certificate merely one of a number of examples which illustrate this relationship. The knowledge and technique of forensic science help in solving cases of crimes, legal disputes, etc. This also helps in solving the problem of crimes and disputes for the identification of victims in cases of murder, accidents, etc., criminals in case of rape, murder, etc. and the father in case of paternity disputes. The scope of forensic medicine is boundless (Honda et al., 1990). The duties of the investigators include finding out the cause of death in suspected cases of homicide, infanticide, suicide and accidental deaths, to provide expert evidence on injuries regarding their manner of causation, their age and their ability to cause death or disability, to help the police in establishing identity of criminals and victims of crime with the help of medical knowledge to establish the time of death in medico legal deaths, to establish proof of foul play or rules it out. The study of the above problems and scientific assembly of evidence for presentation to the legal authorities form the basis of this speciality. All branches of medicine, anatomy, pathology therapeutics and obstetrics provide the basic knowledge and applications of which shapes to conform to the needs of the law to form the body of the subject. The study of the above problems and scientific assembly of evidence for presentation to the legal authorities form the basis of this speciality. The ultimate and sole aim is to arrive at the truth or the nearest approach to it so that justice may prevail on the cases that come up for trial. To attain this objective, an expert in forensic medicine should aim at thoroughness in his investigations, impartial reporting to the concerned authorities and he should depend on scientific truth in the formulation of his findings and conclusions. Through these principles he seeks the constant improvement of the relationship between science and justice in the interest of personal rights, responsibilities and freedoms.

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Proper collection and preservation of the materials used in the scientific experiment will ensure reliability in the results obtained. The next pitfall is in the transmission of material for scientific experiments by specialists in their chosen fields. To make sure that the material is not lost, misplaced or tempered with it is necessary that an unbreakable chain of custody is established. Each person in the chain should take responsibility for the proper transmission of the material to the next person and should maintain a record of the date and time of the recovery of the specimen, what was done with it, when it left possession and to whom given. Needless to mention that the chain should be as short as possible. Scientific observations, often allow more than one interpretation depending upon the circumstances of the situation. It is necessary, therefore, that the investigator is, provided with adequate information of the particular incident, regarding the background and circumstances surrounding the incident, so that, he may draw the correct conclusions from his scientific observations keeping in view the correct perspective of the situation as a whole. Modern scientific investigation is not a one man’s job. It is a team work involving several experts working individually or together with a common aim, namely to arrive at the truth. This concept of team work may be clearly understood if it is agreed that a single incident may present a number of different clues which require the employment of different specialists for their interpretation. Conversely, a single item of evidence may require the combined examination by several specialists. There is now growing recognition of the importance of team work of investigations, particularly in the investigation of murders, mass catastrophes, vehicular accidents, arsons etc. A well trained and experienced forensic pathologist at the scene of crime can be of great assistance to the investigating team to assure that the dead body is properly preserved and handled. Necessary photographs and sketches are made, all potential valuable evidence recognised and collected and marked by appropriate officials. This information will be of great value to the forensic pathologist in his procedures with respect to autopsy of the deceased in determining the cause of death and in forming other opinions concerning how death occurred (accident, suicide and homicide). An autopsy report gives vital clues about the nature of crime and helps in solving the case. Though all doctors are qualified to perform autopsies, experts say vital clues can be missed by them. A forensic medicine expert can pick up fine details during autopsy, which can be missed otherwise. The time, cause and manner of death can be identified during autopsy. Though the aim and object of the system of justice is to discover the facts and to ensure that truth has its full sway on the judgements passed in the courts of law, it is not able to adhere to this aim and object because of some inherent shortcomings in the system. Though the aim of the court is to place responsibility upon the contending parties, in a litigation to present relevant facts and hopefully determine the truth, in reality, the adversary system often undermines the pursuit of truth with opposing sides seeking to win, at all costs, without obligation to reveal information which may be detrimental to them. The lawyer aims at winning in the fight, not at aiding the court to discover the facts. He does not want the trial court to reach a sound and educated guess if it

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is likely to be contrary to his scientific investigation and this will only add to the confusion and may even lead to miscarriage of justice. The quality and reliability of scientific investigations depends on many factors. Failure to take notice of these factors may result in client’s interest. This is a special privilege enjoyed by lawyers. An expert witness has no such privilege and he has no axe to grind. Nor does he have a personal interest to protect a client. His evidence, therefore, is objective, unbiased and impartial as he is not concerned with the outcome of the judgement. A medical witness is not expected to take more responsibility upon himself that he can cope with. Contrary to general belief, forensic medicine is not a static science. It is vital and is continuously growing, taking into its fold any new scientific discovery and effectively turn it to its own use. Many a scientific principle such as photography, neutron activation analysis, Coombs test, etc. has, from time to time, found a permanent and accepted place as significant armaments in the hands of the forensic pathologist in his relentless war against social injustice, misdemeanour and felony.

25.3 Forensic Medicine and DNA Fingerprinting DNA fingerprinting without doubt represents one of the most significant advances in forensic medicine in this century. Some of the commonly used presumptive test reagents for identification of blood and semen could potentially affect the recovery of intact high-molecular-weight deoxyribonucleic acid (DNA) from evidentiary samples (Semba et al., 1994). DNA can be used not only for convicting the guilty but also for exonerating the innocent. It was demonstrated for the first time that a DNA fingerprint could be used to find a perpetrator from within a population. In 1985, a year after the development of DNA fingerprinting, the polymerase chain reaction (PCR) was discovered (Saiki et al., 1985). This discovery has revolutionize the field of molecular biology, though the method would not come into routine use in forensic cases until the early 1990s, since new platforms and biochemical tools were needed in order to take full advantage of the potential of PCR. In particular, new automation technology is the key, and the advent of the automated fluorescent DNA sequencer in the early 1990s is a major step forward. More generally, forensic DNA analysis has benefited substantially from the Human Genome Project, for the genome could be sequenced only with automated equipment that permitted highthroughput processing. Because forensic science could use the same equipment and biochemical tools that gene sequencing used, new methods were rapidly developed in the early 1990s that would have been considered impossible just a few years earlier. Perhaps the best example of this adjunct benefit of genomics is the development of national DNA databases. Since its inception in 1995, the National DNA Database for England and Wales has expanded to include more than 2.75 million reference DNA profiles, against which all specimens obtained from the scene of a crime (“crime stains”) are routinely compared (Werrett et al., 1982). The likelihood that a match will be found is approximately 30%. Many other countries have since

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followed suit, and the benefits of such databases are considerable, since persons who commit serious crimes such as murder usually have a previous criminal record. The United Kingdom’s policy permits the collection of DNA profiles from all convicted criminals, as well as from anyone suspected of committing a crime that could lead to a prison sentence and the law allows authorities to retain the DNA profile even if the suspect is found innocent. Consequently, persons who later commit more crimes can be identified and apprehended quickly (Gill et al., 2000; Marchi, 2004). The analysis of DNA isolated from forensic biological evidence provides valuable information relating to the identification of the source of the sample. Sex determination from blood and bloodstains by polymerase chain reaction (PCR) was also performed (Semba et al., 1994) from the viewpoint of forensic medicine (Fig. 25.1). In a typical case where PCR method was used, Y chromosome specific sequence (DYZ3) and X chromosome specific sequence (DXZ1) belonging to alphoid (alpha) centromeric repeat family were specifically amplified. The limit of detection of the specific sequences by PCR corresponds to 0.00001 μl of the whole blood. In case of diluted blood, it was possible to detect X and Y specific sequences in the specimen. diluted up to 100,000 times. X and Y specific sequences

Fig 25.1 Method for analysis of DNA from forensic samples

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could be detected from a cotton cloth in size of 1 mm2 , on which blood diluted by 10,000 times had been attached. In case of bloodstains, X and Y specific sequences could be detected even from 1 mm of a single cotton fiber and sex could be determined. X and Y specific sequences could be detected even from blood specimen left at room temperature for 7 months, and from bloodstains left at room temperature for two years. Further, sex determination could be achieved from aged bloodstains preserved at room temperature for 22 years. X and Y specific sequences could be distinctly detected after blood specimen was heated in a water bath at 100 degrees C for 9 h. After bloodstains were heated in an electric furnace at 150 degrees C for 30 min, both specific sequences could be detected. When male and female blood specimens were mixed together, X and Y specific sequences were amplified satisfactorily when male-female mixing ratio was from 100:1 to 1:1. From the mixing ratio of 1:10, amplified band of Y specific sequence began to gradually weaken, and only weak band was detected when the mixing ratio was 1:1000. These results reveal that the present sex determination method by PCR can be performed in simple and quick manner and has high detecting sensitivity, and sex can be determined even from putrefied, heated or aged specimens. Thus, it is expected to be one of useful examination methods in forensic practices. RFLPs and 9-bp deletion type of mtDNA may suggest the characteristics of the human races (Misawa, 1994). In a study sex determination using polymerase chain reaction (PCR) on tooth material was evaluated from the viewpoint of forensic medicine by Murakami et al. (2000). Using the method of DNA analysis, Restriction fragment length polymorphism testing, using a combination of single locus probe (SLP) that vary highly among individuals, produces a DNA fingerprint or profile. The PCR method using STR (microsatellite) and mitochondrial DNA analysis (mtDNA) is suitable for examination of the forensic biological samples (bloodstains, hairs, seminal stains, bones, tooth). For sex identification of bloodstains, bleached skeletons and teeth, Southern blot hybridization with Y-chromosome specific probe (pHY10; 3.4 kb) and PCR amplifying with sex chromosome specific fragments, can be used. Mitochondrial DNA from bones and teeth can also help in sex determination (Harihara et al., 1990). The antemortem dental information including dental records and radiological records provided by families of the victims, play a major role in speedy identification (Yamamoto, 1996). Central to this technology, which is based on the analysis of the genetic component of cells, is the use of DNA probes to regions of the human genome that exhibit great variability between individuals. These probes fall into two main categories. The first group comprises those that can detect a large number of these “hyper variable” loci simultaneously, namely multilocus probes (MLPs). On autoradiography, these give rise to a band pattern that is reminiscent of the bar code on supermarket goods, the main advantage of which is that a single such test provides a lot of information very rapidly. MLPs are, therefore, the probes of choice when the amount of material for testing is not limiting, e.g., a blood sample for paternity testing. In many forensic cases, however, the material evidence available for testing is minute, such as a few hair roots or a tiny semen stain, and the situation is often complicated by the presence of tissue from more than one person.

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25.4 Applications of DNA Methylation Markers in Forensic Medicine The methylation pattern of human genome is space-time specific, sex-specific, parent-of-origin specific and disease specific, providing us an alternative way to solve forensic problems (Zhao and Yang, 2005). DNA methylation profiles represent a more chemically and biologically stable source of molecular diagnostic information than RNA or most proteins. Recent advances attest to the great promise of DNA methylation markers as powerful future tools in the clinic. DNA methylation is a post-replication modification that is predominantly found in cytosines of the dinucleotide sequence CpG. Epigenetic information is stored in the distribution of the modified base 5-methylcytosine. In the past decade, DNA methylation analysis has been revolutionized by two technological advances viz. bisulphite modification of DNA and methylation-specific polymerase chain reaction (MSP).

25.5 Forensic Medicine and Anthropometry Forensic medicine is an interdisciplinary science which in everyday practice applies all the knowledge that medical sciences, have accepted as reliable and scientifically solid facts or processes, and qualitative and quantitative definitions with the help of which accurate and reliable statements can be made. The use of anthropometry in the field of forensic science and medicine dates back to 1882 when Alphonse Bertillon, a French police expert invented a system of criminal identification based on anthropometric measurements. His system was based on three fundamental ideas- the fixed condition of the bone system from the age of twenty till death; the extreme diversity of dimensions present in the skeleton of one individual compared to those in another; the ease and relative precision with which certain dimensions of the bone structure of a living person can be measured using simply constructed calipers. This system of identification spread rapidly through much of the world but the system was not accepted much in view of some major drawbacks and discovery of other identification systems e.g. dactylography. As anthropometry is an important part of biological/physical anthropology, hence the persons specializing in anthropometry are familiar with range of biological variability present in the human populations and its causes, and are well trained in comparative osteology, human osteology, craniometry, osteometry, racial morphology, skeletal anatomy and function. They are well aware of the knowledge of archaeological field techniques and methods which serve well in crime scene recoveries involving buried and surface remains. The term “forensic anthropometry” can be coined for this branch of applied physical anthropology, involving the use of methods/techniques of anthropometry in forensic/legal context. In other words, “forensic anthropometry is a scientific specialization emerged from the discipline of forensic anthropology dealing with identification of human remains with the help of metric techniques”.

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Anthropometric characteristics have direct relationship with sex, shape and form of an individual and these factors are intimately linked with each other and are manifestation of the internal structure and tissue components which in turn, are influenced by environmental and genetic factors. Anthropometric data are believed to be objective and they allow the forensic examiner to go beyond subjective assessments such as “similar” or “different”. With measurement data, the examiner is able to quantify the degree of difference or similarity and state how much confidence can be placed in this interpretation. The main aim of an anthropometrist employed in the forensic medicine/medicolegal department, working with unknown variables, is to describe the remains in such terms so that one can achieve the goal of estimating age at the time of death, sex, stock/race/ancestry/ethnicity, stature, body weight/body build, details of individualizing characteristics i.e. amputations, fractures, ankyloses, deformities and bone pathologies and to some extent the cause of death if reflected in the remains/bones. The objective is to enable the law enforcement agencies to achieve the ultimate goal of personal identification. Forensic anthropometry incorporates most of the techniques originating with the analysis of human skeletal material from archaeological sites; the two disciplines have been closely linked. A good forensic anthropologist must, by definition, be a good skeletal biologist. He helps a forensic pathologist to reconstruct the biological nature of the individual at the time of postmortem examination, and sometimes giving clues and reconstructing the circumstances surrounding death. He is prepared for this by his training in describing the prehistoric skeletons from archaeological sites and usually by special experience in identifying unknown modern skeletons.

25.6 Clinical Forensic Medicine Clinical forensic medicine (CFM) is “the application of appropriate forensic practices and principles, reserved for use by the pathologist at autopsy, to living patients in a clinical setting.” “Living forensic” patients include survivors of trauma and potentially catastrophic experiences resulting in injury. CFM arose from “clinically” affirming that not all abuse or assault victims sustain fatal injuries. Appropriate medical documentation and interpretation of physical findings may aid law enforcement and/or social services in the legal evaluation of a case or situation. Additionally, timely collection of pertinent evidence may be performed as the case necessitates. (Recktenwald et al., 2005) Criminal violence and its associated trauma comprise a critical health problem throughout the world. Clinical forensic medicine represents a new discipline of medical practice that is evolving in direct response to the sequelae of criminal and interpersonal violence. The application of the principles and standards of the forensic specialist has been increasingly recognized as playing a crucial role in trauma care; the results of the extremes of human behaviour-abused children, individuals suffering from blatant neglect and maltreatment, or self-inflicted injury, and

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victims of road-traffic accidents, firearm injuries and other assaults. These cases must be reported to a legal agency for investigation and follow-up. As trends in crime and violence change, new antiviolence legislation is likely to be implemented; consequently, new personnel resources are required to ensure that these legislative mandates effectively meet the needs of society (Sharma, 2006). Recently it was reported that post-mortem histopathological investigations of the bone marrow in forensic medicine is an important issue for both the forensic and clinical pathologist (Roll et al., 2009).

25.7 Conclusion The technological advancement in forensic medicine will be imposing challenges, including those concerning genetic manipulation and the keeping of confidentiality regarding electronic files. On the other hand, old problems will recur, the most important being the vital question of research with prisoners and the use of torture by agents of the state. There is a need for international legislative mechanisms that would establish a set of explicit rules of behavior, thus diminishing the possibility of ethical dilemmas. The subject of forensic medicine has grown from the days when it was taught as part of pathology, to being a specialty in itself. However, forensic medicine has to advance to keep pace with new developments and we need the committed efforts of all doctors in this profession to meet the expectations of society.

References Gill, P., Whitaker, J., Flaxman, C., Brown, N., Buckleton, J., 2000, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int 112:17–40. Harihara, S., Nakanura T., Hirai, M., et al., 1990, Sex indentification by analysis of DNA extracted from hard tissues. Nihon Hoigaku Zasshi 44(4):293–301. Honda, K., Harihara, S., Nakamura, T., Hirai, M., Misawa, S., 1990, Sex identification by analysis of DNA extracted from hard tissues. Nihon Hoigaku Zasshi 44:293–301. Marchi, E., 2004, Methods developed to identify victims of the World Trade Center disaster. Am Lab 36:30–36. Misawa, S., 1994, Application of DNA polymorphism to forensic medicine. Rinsho Byori 42: 636–642. Murakami, H., Yamamoto, Y., Yoshitome, K., Ono, T., Okamoto, O., Shigeta, Y., Doi, Y., Miyaishi, S., Ishizu, H., 2000, Forensic study of sex determination using PCR on teeth samples. Acta Med Okayama 54:21–32. Recktenwald, K., Hunsaker, D.M., Corey, T.S., Weakley-Jones, B., 2005, Clinical forensic medicine introduction for healthcare providers. J Ky Med Assoc 103:433–435. Roll, P., Beham, A., Beham-Schmid, Ch., 2009, Post-mortem histopathological investigations of the bone marrow in forensic medicine: an important issue for both the forensic and clinical pathologist. Forensic Sci Int 186:e17–e20. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., Arnheim, N., 1985, Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354.

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Semba, S., Yamamoto, Y., Ishizu, H., 1994, Sex determination from blood and bloodstains by polymerase chain reaction (PCR). Nihon Hoigaku Zasshi 48(1):7–18. Sharma, B.R., 2006, Clinical forensic medicine in the present day trauma-care system-an overview. Injury 37:595–601. Werrett, D., Pinchin, R., Hale, R., et al., 1982, Problem solving: DNA data acquisition and analysis. Prof DNA 2:1–6. Werrett, D., Pinchin, R., Hale, R., 1982, Problem solving: DNA data acquisition and analysis. Prof DNA 2:1–6. Yamamoto, K., 1996, Molecular biological studies on teeth, and inquests. Forensic Sci Int 80: 79–87. Zhao, G.S., Yang, Q.E., 2005, Applications of DNA methylation markers in forensic medicine. Fa Yi Xue Za Zhi 21:61–64.

Chapter 26

Pharmacogenomics

Abstract Pharmacogenomics explores the contribution of genetics to drug safety and specifically examines the single gene interactions with drugs. It also makes use of genetic tests to distinguish among patients whose genetic characteristics predispose them to respond in certain ways to certain medicines. An understanding of the genetic variables that influence drug response could also help pharmaceutical companies design new, more effective therapies. Keywords Drugs · Pharmacogenomics · Drug response · Gene variation · Personalised medicine · SNP · Drug discovery · Pharmacogenomic tests

26.1 Prologue Revolutionary new technologies, capable of transforming the economics of sequencing, are providing an unparalleled opportunity to analyze human genetic variation comprehensively at the whole-genome level within a realistic timeframe and at affordable costs. Current estimates suggest that it would cost somewhere in the region of US$30 million to sequence an entire human genome using Sanger-based sequencing, and on one machine it would take about 60 years. When applied over a large enough genomic region, these new approaches to resequencing will enable the simultaneous detection and typing of known, as well as unknown, polymorphisms, and will also offer information about patterns of linkage disequilibrium in the population being studied (Shi and Chen, 2002). Technological progress, leading to the advent of single-molecule-based approaches, is beginning to dramatically drive down costs and increase throughput to unprecedented levels, each being several orders of magnitude better than that which is currently available (Lee, 2009). A new sequencing paradigm based on single molecules will be faster, cheaper and more sensitive, and will permit routine analysis at the whole-genome level (Johnson and Evans, 2002).


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26.2 Concept Despite advances in modern medicine, in practice it remains difficult for physicians to determine a patient’s response to a treatment. A drug that is safe and effective for one patient may prove to be dangerous or ineffective in another. In some cases, the lack of effectiveness of a drug or the occurrence of side-effects can be linked to factors such as medication errors. However, it is now known that a significant proportion of differential drug response is genetic in origin (López-López et al., 2004). This genetic angle has often not been fully appreciated, but new developments are driving the use of this information in medicine. Pharmacogenomics is the study of how an individual’s genetic inheritance affects the body’s response to drugs (Evans and Relling, 1999). The term comes from the words pharmacology and genomics and is thus the intersection of pharmaceuticals and genetics. Pharmacogenomics refers to the general study of all of the many different genes that determine drug behaviour. The way a person responds to a drug (this includes both positive and negative reactions) is a complex trait that is influenced by many different genes. Without knowing all of the genes involved in drug response, scientists have found it difficult to develop genetic tests that could predict a person’s response to a particular drug. Scientists discovered that genes show small variations (or changes) in their nucleotide (DNA base) content. Genetic testing for predicting drug response is now possible. The inherited variations in genes that dictate drug response and explores the ways these variations can be used to predict whether a patient will have a good response to a drug, a bad response to a drug, or no response at all .This study also allows to know the influence of genetic variation on drug response in patients by correlating gene expression or single-nucleotide polymorphisms with a drug’s efficacy or toxicity (Johnson and Evans, 2001). By doing so, pharmacogenomics aims to develop rational means to optimise drug therapy, with respect to the patients’ genotype, to ensure maximum efficacy with minimal adverse effects. Pharmacogenomics holds the promise that drugs might one day be tailor-made for individuals and adapted to each person’s own genetic makeup (Evans and Relling, 1999). Environment, diet, age, lifestyle, and state of health all can influence a person’s response to medicines, but understanding an individual’s genetic makeup is thought to be the key to creating personalized drugs with greater efficacy and safety (Mancinelli et al., 2000). Pharmacogenomics combines traditional pharmaceutical sciences (Fig. 26.1) such as biochemistry with annotated knowledge of genes, proteins, and single nucleotide polymorphisms (Blake and Sobel, 2008). Pharmacogenomics seeks to uncover significant associations between genomic patterns and clinical outcomes. It also helps to produce useful predictive knowledge, allowing clinical treatment decision making to be based upon more rational criteria than today’s probabilistic approach, which is largely based upon educated guesswork. Most of the attention on pharmacogenomics has focused on its role in bringing future drugs to market, but the technology could also be used to identify genetic risk factors for adverse drug reactions associated with drugs.

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Fig 26.1 Pharmacogenomics, a key to creating personalized drugs

26.3 Predicting Drug Response on Gene Variation DNA sequencing is the determination of the order of nucleotides (the base sequence) in a DNA molecule. As many of the genetic variations found within the human genome as possible. These variations or SNPs can be used as a diagnostic tool to predict a person’s drug response. For SNPs to be used in this way, a person’s DNA must be examined (sequenced) for the presence of specific SNPs. The problem is, however, that traditional gene sequencing technology is very slow and expensive and has therefore impeded the widespread use of SNPs as a diagnostic tool (Dan et al., 2006). DNA microarrays (or DNA chips) are an evolving technology that should make it possible for doctors to examine their patients for the presence of specific SNPs quickly and affordably. A single microarray can now be used to screen 100,000 SNPs found in a patient’s genome in a matter of hours. As DNA microarray technology is developed further, SNP screening in the doctor’s office to determine a patient’s response to a drug, prior to drug prescription, will be commonplace.

26.4 Drug Development and Testing Benefit from Pharmacogenomics Pre-screening should allow clinical trials to be smaller, faster, and less expensive; therefore, the consumer could benefit in reduced drug costs. SNP screenings will benefit drug development and testing because pharmaceutical companies could exclude from clinical trials those people whose pharmacogenomic screening would show that the drug being tested would be harmful or ineffective for them. Excluding

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these people will increase the chance that a drug will show it useful to a particular population group and will thus increase the chance that the same drug will make it into the marketplace. Pre-screening clinical trial subjects should also allow the clinical trials to be smaller, faster, and therefore less expensive; therefore, the consumer could benefit in reduced drug costs. Finally, the ability to assess an individual’s reaction to a drug before it is prescribed will increase a physician’s confidence in prescribing the drug and the patient’s confidence in taking the drug, which in turn should encourage the development of new drugs tested in a like manner.

26.5 Applications and Benefits of Pharmacogenomics Pharmaceutical companies will be able to create drugs based on the proteins, enzymes, and RNA molecules associated with genes and diseases. This will facilitate drug discovery and allow drug makers to produce a therapy more targeted to specific diseases. This accuracy not only will maximize therapeutic effects but also decrease damage to nearby healthy cells (Evans and Relling, 1999).

26.5.1 Better, Safer Drugs the First Time Instead of the standard trial-and-error method of matching patients with the right drugs, doctors will be able to analyze a patient’s genetic profile and prescribe the best available drug therapy from the beginning. Not only will this take the guesswork out of finding the right drug, it will speed recovery time and increase safety as the likelihood of adverse reactions is eliminated. Pharmacogenomics has the potential to dramatically reduce the estimated 100,000 deaths and 2 million hospitalizations that occur each year in the United States as the result of adverse drug response.

26.5.2 More Accurate Methods of Determining Appropriate Drug Dosages Current methods of dosages on weight and age will be replaced with dosages based on a person’s genetics, how well the body processes the medicine and the time it takes to metabolize it. This will maximize the therapy’s value and decrease the likelihood of overdose. • Advanced Screening for Disease Knowing one’s genetic code will allow a person to make adequate lifestyle and environmental changes at an early age so as to avoid or lessen the severity of a genetic disease. Likewise, advance knowledge of particular disease susceptibility will allow careful monitoring, and treatments can be introduced at the most appropriate stage to maximize their therapy.

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• Better Vaccines Vaccines made of genetic material, either DNA or RNA; promise all the benefits of existing vaccines without all the risks. They will activate the immune system but will be unable to cause infections. They will be inexpensive, stable, easy to store, and capable of being engineered to carry several strains of a pathogen at once. • Improvements in the Drug Discovery and Approval Process Pharmaceutical companies will be able to discover potential therapies more easily using genome targets. Previously failed drug candidates may be revived as they are matched with the niche population they serve. The drug approval process should be facilitated as trials are targeted for specific genetic population groups providing greater degrees of success. The cost and risk of clinical trials will be reduced by targeting only those persons capable of responding to a drug. • Decrease in the Overall Cost of Health Care Decreases in the number of adverse drug reactions, the number of failed drug trials, the time it takes to get a drug approved, the length of time patients are on medication, the number of medications patients must take to find an effective therapy, the effects of a disease on the body (through early detection), and an increase in the range of possible drug targets will promote a net decrease in the cost of health care.

26.6 Benefits of Pharmacogenomic Testing As with other types of genetic testing, pharmacogenomic tests usually require only a small sample, such as blood or a scraping from inside the cheek. Results would let the physician predict whether someone will respond positively to drug therapy within hours rather than in the days or weeks it might take with the trial-and-error method and with substantially less risk to the patient. In the end, these tests may give doctors invaluable information about their patients not otherwise available to them (López-López et al., 2004).

26.6.1 Patient’s Ability to Metabolize Drugs Testing patients prior to initiating drug therapy to determine their ability to metabolize different classes of drugs is a key emerging area of investigation. Such metabolic information could prove useful to both the doctor and patient when choosing current and future drug therapies and drug doses. There are a number of types of enzymes in the liver that metabolize medications. Genetic variations in these enzymes that affect metabolic rate are relatively common, but the prevalence of the variations differs significantly by ethnic background. Some of these enzymes include: • • • •

The Cytochrome P450 family N-acetyltransferase Thiopurine methyltransferase (TPMT) UDP-glucuronosyltransferase

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The Cytochrome P450 family: Some of the most studied enzymes are the members of the Cytochrome P450 (CYP) family of about 50 liver enzymes. These enzymes metabolize more than 30 classes of drugs, including antidepressants, antiepileptics and cardiovascular drugs. Patients can be separated into poor, normal and ultra-rapid metabolizers of drugs by the CYP enzymes. These classifications are due to variations in the associated CYP gene. When a poor metabolizer of a particular drug is given a standard dose of that drug, he will process the drug more slowly, resulting in increased levels of the drug in his bloodstream, the potential for side effects, and an increased risk of toxicity. For an ultra-rapid metabolizer, the same dose may be ineffective as the drug is processed too rapidly to have its full effect. Dosages of these drugs must be altered to accommodate the rate of metabolism. The CYP family is important as it affects the metabolism of a significant percentage of available drugs and because a significant proportion of the population are poor or ultra-rapid metabolizes (Kirchheiner and Seeringer, 2007; Kirchheiner et al., 2005). N-acetyltransferase: This is a liver enzyme that activates some drugs and deactivates others. Some patients can acetylate (a type of metabolic change) drugs slowly while other patients acetylate drugs quickly. Those who are slow acetylators may experience toxicity when taking drugs such as procainamide, isoniazid, hydralazine, and sulfonamides. Those who are fast acetylators may not respond to isoniazid or hydralazine. About 40–70% of Caucasians and African-Americans are considered slow acetylators. Thiopurine methyltransferase (TPMT): This enzyme metabolizes the immune suppressant azathioprine and other thiopurine medications such as 6-mercaptopurine and 6-thioguanine (used to treat children with acute lymphocytic leukemia and also used to treat autoimmune diseases). Each copy of the TPMT gene will produce some TPMT enzyme. This leads to three different groups of enzyme activity levels (low/low, low/high, and high/high or deficient, intermediate, and normal). About 1 in 300 Caucasians and African-Americans are TPMT- deficient. If these patients are given a standard drug dose, they may suffer severe hematopoietic (red blood cell producing) toxicity. Many are able to achieve the desired therapeutic effect from a dose that is one tenth of the “normal” dose. UDP-glucuronosyltransferase: This enzyme is involved in the metabolism of the chemotherapy drug irinotecan, which is used in the treatment of metastatic colorectal cancer. Variation in the gene that code for this enzyme can influence the patient’s ability to break down the major active metabolite. The inability to break the metabolite down can lead to increased levels of it in the blood and a higher risk of side effects, which include reduced white blood cell count and severe diarrhea.

26.6.2 Age-Related Genetic Variations Some researchers are looking at changes in genetic variation over time to help evaluate how age may affect genetic response to drug therapy.

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26.7 Recent Reports of Pharmacogenomics Use For many drugs, pharmacogenetic polymorphisms are known affecting biotransformation and clinical outcome. The clinical importance of these variants depends on allele-frequency and the effect size of the clinical outcome parameters. Further, it depends on the therapeutic range of the drug which is affected, on predictability of drug response as well as on duration until onset of therapeutic efficacy. Consequences which arise from genotyping might be: adjustment of dose according to genotype, choice of therapeutic strategy or even choice of drug. In antidepressant drug treatment, most drugs are metabolized via the polymorphic cytochrome P450 enzyme CYP2D6. Huge differences in pharmacokinetic parameters have been consistently shown for many tricyclics, some SSRIs, and other antidepressant drugs whereas the effects on therapeutic efficacy and adverse events have been described controversially. In cardiovascular disease, oral anticoagulants, nonsteroidal anti-inflammatory drugs, oral hypoglycemic drugs and other drugs are affected by genetic polymorphisms of the cytochrome P450 drug metabolizing enzyme CYP2C9. Studies in patients or healthy volunteers revealed up to 10-fold differences in pharmacokinetic parameters due to genetic polymorphisms of CYP2C9. Pharmacogenetics based dose adjustments are one tool to individualize drug treatment according to genetic factors. They can be derived from pharmacokinetic data with the aim to obtain equal drug concentrations in each individual. Prospective validation of dose adjustments based on pharmacogenetics should be performed before routine application of such strategies. A controlled prospective clinical trial with one arm receiving genotype-based dose adjustments and the other arm receiving therapy as usual will elucidate the benefit of pharmacogenomics-based individualization of certain drug therapies. Pharmacogenetic variability plays an important role in the pharmacokinetics of oral antidiabetic drugs; however, to date, the impact of this variability on clinical outcomes in patients is mostly unknown and prospective studies on the medical benefit of CYP genotyping are required (Kirchheiner et al., 2006). Loss of function of thiopurine S-methyltransferase (TPMT) results in severe and life-threatening hematopoietic toxicity if patients receive standard doses of mercaptopurine and azathioprine. Gene duplication of cytochrome P4502D6 (CYP2D6), which metabolizes many antidepressants, has been identified as a mechanism of poor response in the treatment of depression. There is also a growing list of genetic polymorphisms in drug targets that have been shown to influence drug response. A major limitation that has moderated the use of pharmacogenetic testing in the clinical setting is the lack of prospective clinical trials demonstrating that such testing can improve the benefit/risk ratio of drug therapy. As research has graduated from studying single candidate genes to wholegenome scans, pharmacogenomics is beginning to make its impact on the therapeutics of complex CNS disorders, such as schizophrenia, Parkinson’s disease and Alzheimer’s disease. Alzheimer’s disease is a progressive complex disorder, where genetic predisposition interacts with environmental factors. With conventional therapeutics only providing symptomatic treatment, the current focus of the

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pharmaceutical industry is on novel strategies with an etiopathogenic orientation (Gupta et al., 2008). Today, clinical trials researchers use genetic tests for variations in cytochrome P450 genes to screen and monitor patients. In addition, many pharmaceutical companies screen their chemical compounds to see how well they are broken down by variant forms of CYP enzymes (Kirchheiner and Seeringer, 2007). Another enzyme called TPMT (thiopurine methyltransferase) plays an important role in the chemotherapy treatment of a common childhood leukemia by breaking down a class of therapeutic compounds called thiopurines. A small percentage of Caucasians have genetic variants that prevent them from producing an active form of this protein. As a result, thiopurines elevate to toxic levels in the patient because the inactive form of TMPT is unable to break down the drug. Today, doctors can use a genetic test to screen patients for this deficiency, and the TMPT activity is monitored to determine appropriate thiopurine dosage levels. Pharmacogenomics can help in translating this knowledge of human genome variability into efficacious and safer therapeutics. In another research the potential contributions of pharmacogenomics to improved treatment in the 21st century in context to Brain cancer. Patients with oligodendrogliomas have benefited from phamacogenomics, as there is a clear relationship between response to chemotherapy and chromosomal profile. Drug efficacy, safety and response could be improved by using pharmacogenomics to identify genetic markers that differentiate responder from nonresponder patient groups, as well as identifying patients likely to develop adverse drug reactions (Shai et al., 2008). Thus, pharmacogenomics by microarray studies may lead to much more accurate tumor classification, drug and biomarker discovery, and drug efficacy testing.

26.8 Interpreting Pharmacogenomic Tests Pharmacogenomic test results can be difficult to interpret. There are limitations to interpretation because enzymes involved in drug metabolism arise from multiple genes and that process is often complex. The test results are predictions based on information about the specific genetic variations and on information about the associated diseases, adverse drug reactions, and patient outcomes that have been gathered during studies and clinical trials. In many cases, the predictions will be very accurate, but they cannot say with 100% certainty what will happen with an individual patient and they do not incorporate or make allowances for the other factors in a patient’s life related to the disease condition or to the individual that may also affect their response to treatment. This is one of the reasons why the results are intended to be used in conjunction with other relevant clinical findings. Some currently available pharmacogenomic tests include: • A DNA microarray that tests for 29 CYP2D6 genetic variants and 2 for the CYP2C19. It is meant to be used as an aid in individualizing treatment selection and dosing for drugs metabolized through these genes. It helps predict poor, intermediate, extensive, or ultra-rapid metabolizers.

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Barriers to Pharmacogenomics Progress

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• A test that detects variations in the UGT1A1 gene, which produces the enzyme UDP-glucuronosyltransferase. The enzyme is active in the metabolism of drugs such as irinotecan, a drug used in metastatic colorectal cancer treatment. The test is used to identify patients who may be at increased risk of adverse reaction to the drug. The gene that the test detects has been shown to be an effective genetic marker for predicting irinotecan-induced toxicity. • Tests that detect genetic variants of the CYP2C9 and VKORC1 (vitamin K epoxide reductase) enzymes. These enzymes are involved in the efficacy of warfarin as an anticoagulant. Warfarin is used to prevent dangerous blood clots from forming in the blood vessels of certain patients, but it can significantly increase the risk of bleeding into the head or gastrointestinal tract. These tests identify patients who have genetic variations and so need a reduced dose of warfarin to avoid bleeding episodes. An FDA advisory committee voted in November 2005 in favor of changing Warfarin’s label to reflect the fact that pharmacogenomic information can be useful in deciding a patient’s individual dose. Pharmacogenomic tests are promising but there are questions and concerns about their utility, use, interpretation, ability to predict actual drug response, their effect on patient outcome, and about the potential for the results of testing to be used to discriminate against the patient or to prevent them from access to treatment.

26.9 Barriers to Pharmacogenomics Progress Pharmacogenomics is a developing research field that is still in its infancy. Several of the following barriers will have to be overcome before many pharmacogenomics benefits can be realized. • Complexity of finding gene variations that affect drug response – Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered. SNPs occur every 100–300 bases along the 3-billion-base human genome, therefore millions of SNPs must be identified and analyzed to determine their involvement (if any) in drug response. Further complicating the process is our limited knowledge of which genes are involved with each drug response. Since many genes are likely to influence responses, obtaining the big picture on the impact of gene variations is highly time-consuming and complicated. • Limited drug alternatives – Only one or two approved drugs may be available for treatment of a particular condition. If patients have gene variations that prevent them using these drugs, they may be left without any alternatives for treatment. • Disincentives for drug companies to make multiple pharmacogenomic products – Most pharmaceutical companies have been successful with their “one size fits all” approach to drug development. Since it costs hundreds of millions of dollars to bring a drug to market, will these companies be willing to develop alternative drugs that serve only a small portion of the population?

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• Educating healthcare providers – Introducing multiple pharmacogenomic products to treat the same condition for different population subsets undoubtedly will complicate the process of prescribing and dispensing drugs. Physicians must execute an extra diagnostic step to determine which drug is best suited to each patient. To interpret the diagnostic accurately and recommend the best course of treatment for each patient, all prescribing physicians, regardless of specialty, will need a better understanding of genetics.

26.10 The Promise of Pharmacogenomics Right now, in doctors’ offices all over the world, patients are given medications that either don’t work or have bad side effects. Often, a patient must return to their doctor over and over again until the doctor can find a drug that is right for them. Pharmacogenomics offers a very appealing alternative. Imagine a day when you go into your doctor’s office and, after a simple and rapid test of your DNA, your doctor changes her/his mind about a drug considered for you because your genetic test indicates that you could suffer a severe negative reaction to the medication. However, upon further examination of your test results, your doctor finds that you would benefit greatly from a new drug on the market, and that there would be little likelihood that you would react negatively to it. A day like this will be coming to your doctor’s office soon, brought to you by pharmacogenomics (Philips et al., 2000). Although pharmacogenomics has the potential to radically change the way health care is provided, it is only in its infancy. In the future, pharmacogenomics could find uses along the entire drug discovery and development timeline, all the way from target discovery and validation to late-stage clinical trials (Fig. 26.2) Beyond that, pharmacogenomic tests could find their way into the doctor’s office as a means to get the right medicine to the right patient at the right time (Terra and Johnson, 2002). While genetics and genomics are often used synonymously, pharmacogenetics is more focused in scope than and is viewed as a subset of pharmacogenomics, which encompasses factors beyond those that are inherited. Some people believe that pharmacogenomics will lead to the stratification of diseases into genetically defined categories. For pharmacogenetics to be effective, markers must be found that

Fig. 26.2 Promises of pharmacogenomics: a new drug for a individualised therapy

26.10

The Promise of Pharmacogenomics

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indicate the connection between drug response and genetic makeup. The markers that companies are pursuing most diligently are known as single-nucleotide polymorphisms, or SNPs. SNPs, which are defined only in relation to a population, are variations in DNA at a single base that are found in at least 1% of the population (Tolle, 2001). Thinking that breast cancer markers such as BRCA1 and BRCA2 only provide information about susceptibility. Obviously, breast cancer caused by BRCA1 is a different kind of cancer than cancers not caused by BRCA1 mutations. Pharmacogenomics can be used in many ways during drug discovery and development. For example, it can identify variations in drug targets and ensure that companies are screening against the most common variant. However, pharmacogenomics is most likely to be used during the clinical development process (Krynetski and Evans, 1998). Pharmaceutical companies will be able to use pharmacogenetics to help differentiate their drugs in a crowded marketplace. Meanwhile, search is on for pharmacogenomic markers for osteoarthritis, which encompasses about half of all the cases of arthritis. In this disease, which affects primarily women older than 45, cartilage in the joints breaks down. One of the reasons drugs haven’t been developed is that the progression time is long, so the clinical trials are long. The progression is monitored by taking X-rays of the joints and measuring the thinning of the distance between the bones. Clinical trials are expensive because they require many patients and a long time. Pharmacogenomics will probably be most successful in areas such as oncology (Shai et al., 2008), where many therapies are available but each one works for only a small percentage of patients. Getting the treatment right the first time can be important, even life-saving. If a patient has cancer and is going to be treated with a drug for an extended period before you find out if the drug has worked, and if you know that a given drug is only going to work in 10% of patients, there’s a very strong reason for a physician to do a test to try to get the drug right from day one. Toxicity is another important application of pharmacogenomics. One such test, developed by researchers at St. Jude Children’s Research Hospital in Memphis, identifies patients who have mutations in the thiopurine S-methyltransferase gene, which is involved in the metabolism of mercaptopurine drugs. Children who metabolize these drugs too slowly can literally overdose on their treatment. Technology must also advance if the field is to move forward. A platform that can do large amounts of genotyping inexpensively is required, Right now, the cheapest real way to do genotyping costs about a dollar per SNP. If you want to look at 100,000 SNPs, it’s $100,000 a patient, which just isn’t practical. You need to get that down to a penny [per SNP]. Then suddenly there are a lot of interesting experiments that you can do that you can’t do today. Technology that is inexpensive could be available in as early as three, but no later than 10 years from now, he predicts. In addition to technological improvements, gaps in the knowledge of the human genome need to be filled. Pharmacogenomics is the study of how drugs are metabolized in the body and the variations in the genes that produce the metabolizing enzymes. By studying the genes that produce the specific enzymes that metabolize a drug that is to be

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prescribed, a doctor may decide to raise or lower the dose, or even change to a different drug. The decision about which drug to prescribe may also be influenced by other drugs the patient is taking, to avoid drug-drug interactions. Currently, doctors typically prescribe one of several appropriate drugs for their patients. They prescribe a “standard” dose based on factors such as weight, sex, and age, and then adjust the dose over time, depending on whether the patient’s condition is responding to the medication and whether the patient is experiencing unpleasant or dangerous side effects. The concentrations of ome drugs are monitored with blood tests and the dosages increased or decreased to maintain the drug level in an established “therapeutic” range. Follow-up of such processes is called “Therapeutic Drug Monitoring.” If the drug is not effective in treating or controlling the patient’s condition, then the patient is given a different drug and the process is started again. Instead, pharmacogenomics offers physicians the opportunity to individualize drug therapy for patients based on their genetic make-up. For certain drugs, pharmacogenomics is already helping physicians predetermine dosages to have a better chance of achieving the desired therapeutic effect while reducing the likelihood of adverse effects (Johnson, 2001).

26.11 Impact of Drug Discovery, Development, and Marketing Affected by Pharmacogenomics In the past, drug companies have focused primarily on blockbuster drugs, drugs that could be marketed to a large portion of the general population. Each of these drugs took hundreds of millions of dollars and years of research and clinical testing before it was cleared by the FDA and released to the market. For every blockbuster, there were numerous drugs that were orphaned (shelved) during the development process. Some of these drugs were only effective for a small percentage of those enrolled in clinical trials; others had too many associated side effects and complications (Blake and Sobel, 2008). Each of the blockbusters released was also associated with rare but sometimes serious complications, and some complications that were not evident during clinical trials emerged with long term use. When a large number of people began taking these medications, even a tiny percentage of rare complications could become a significant number of affected patients. In some cases, these blockbusters had to be removed from the market and/or their benefits versus their risks weighed carefully by the patients taking them and the doctors prescribing them. With pharmacogenomics, new drug development could be sped up in a couple of ways. First, the human genome project and advances in the mapping and collecting of specific types of genetic variations like SNPs, haplotypes, and microsatellites, have made it potentially easier for drug companies to identify genes of interest (those in which genetic variation is strongly associated with an identifiable disease) and targets of interest (the functioning of specific enzymes, proteins, or receptors that can be used as drug targets to block, inhibit, replace, or enhance the action of the target and have an effect on the condition).

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Impact of Drug Discovery, Development, and Marketing

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And second, it can make it easier to identify the population most likely to benefit from the drug being developed. The results of phase I and phase II clinical trials (performed for safety and effectiveness) can be evaluated to determine common factors in patients who responded versus those who did not or who had significant side effects. Some of the orphan (shelved) drugs may be able to be reevaluated and retargeted at those most likely to respond. In the future, drugs and the tests used to determine who would be likely to benefit from them may be developed simultaneously. Some patients and their advocates have concerns that pharmacogenomic test results might be used to discriminate against patients who have a genetic risk for health problems. Insurance carriers may mandate testing prior to approving drug therapy or require testing of family members. Patient privacy is an issue as with other genetic tests. These and other issues are currently being debated and addressed by members of the medical community, insurers, government agencies, and a variety of national organizations (Lazarou et al., 1998). Pharmacogenomics is already having an impact in facilities where it is being used to guide drug therapy. As more data are reported, it will become more widely accepted until it is the standard of practice. This will happen on a drug-by-drug basis over the next few years. Some classes of drugs that interest researchers at present and hold promise for the future are those used for post-transplant immunosuppression and targeted tumor therapy. Immunosuppressive medications given to patients after an organ transplant must remain within a narrow range in the blood to help prevent rejection of the transplant without causing significant side effects or toxicity. Testing patients for their likely response to these medications prior to transplant would help determine the right drug(s) and the appropriate dose. With cancer, the patient’s ability to tolerate a drug and the tumor’s response to treatment are both important (Krynetski and Evans, 1998). Pharmacogenomic testing could be performed to determine which drug(s) the patient is likely to be able to metabolize efficiently and at what doses, and the tumor tissue could be tested to determine which treatment options it is likely to respond to. Pharmacogenomics has the potential to aid in the development of drugs that target cancer cells specifically without affecting normal tissues and cells. This could lead to safer and more effective treatment of cancers. Pharmacogenomics will help physicians and their patients by enabling pharmaceutical companies to bring more drugs to market that are targeted at those who are most likely to benefit from them. Ultimately, the goal of pharmacogenomic testing is to help medicine and pharmacotherapy become less uncertain disciplines and more capable of improving the quality of an individual’s life through a more personalized approach to drug therapy. In recognizing that patients respond to drugs differently and in working to individualize drug therapy, pharmacogenomics has become an integral part of personalized medicine (Eichelbaum et al., 2006). The emergence of pharmacogenetics in the twentieth century followed a path forged by advances in molecular biology and genetics (Terra and Johnson, 2002). Before technology allowed the study of individual genetic variation, the field

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concentrated on identifying racial and ethnic variations in response to drugs. Most notable among several landmark studies, perhaps, was University of Toronto Professor emeritus Werner Kalow’s investigation in the 1950s of the occurrence of prolonged paralysis and rare, unexplained deaths in surgical patients receiving succinylcholine, a neuromuscular blocker tolerated well by most patients. Kalow discovered that a genetically based deficiency in the metabolizing enzyme pseudocholinesterase was responsible, and proceeded to describe the population incidence of the various alleles responsible for the deficiency. Similar studies confirmed the genetic basis of the variability, seen in response to a wide variety of drugs. As the wider fields of genetics and molecular biology progressed, so did pharmacogenetics. Now essentially folded into the burgeoning science of pharmacogenomics, the discoveries that have emerged from the progenitor field are today available and in use in the diagnostic arena, helping to screen patients who fall into broad populations that, due to their metabolic genotypes, should not receive specific drugs.

26.12 Pharmacogenomics Initiatives The rapid development of pharmacogenomics has led to an encouraging amount of scientific cooperation and collaboration among government, industry, and academia. Large-scale collaborative initiatives are making significant contributions to the effort to eventually bring the benefits of pharmacogenomics to the bedside. One such effort, the Pharmacogenetics Research Network (PGRN), was established in 2000 and currently funds 13 academic research groups conducting basic research describing pharmacogenetic phenotypes and relating them to genetic and genomic information. The PGRN is spearheaded by the NIGMS, with the participation of the National Cancer Institute, the National Heart, Lung, and Blood Institute, the National Human Genome Research Institute, the National Library of Medicine, and the NIEHS, making it a true trans-NIH effort.

26.13 Pharmacogenomics and the Pharmaceutical Industry The pharmaceutical industry has seen the future. In a 9 April 2003 presentation to a U.S. Food and Drug Administration (FDA) Science Board Advisory Committee meeting, Brian Spear, Director of pharmacogenomics at Abbott Laboratories in Abbott Park, Illinois, put it succinctly: “Pharmacogenomics is not something that a company here and a company there have taken a chance on. This is now a standard part of the drug discovery and development process in every one of the drug discovery and research companies.” The industry’s avid pursuit of pharmacogenomics, as evidenced by a recent spate of acquisitions of pharmacogenomically oriented biotechnology firms by the major pharmaceutical companies, runs the gamut from drug discovery to enhancing the safety and efficacy of drugs that have been on the market for many years. Just as the days of the one-drug-fits-all treatment approach may be numbered, so too may

26.14

Applied Pharmacogenetics

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be those of the present pharmaceutical industry business model, which relies heavily on the periodic introduction of blockbuster drugs (typically defined as products with annual revenues in excess of $1 billion) to generate profits and fund research and development. The hope is that the application of pharmacogenomics and other genomics technologies will enable a new paradigm to emerge, with lower development costs, fewer candidate drug failures, revitalized existing products, the possible resuscitation of withdrawn drugs, and a “portfolio” approach to the introduction of new agents, with drugs available in different formulations to maximize safety and efficacy in specific phenotypic populations. According to a report, it currently takes on average $880 million and 15 years to bring a new drug to market. Failed candidate compounds represent a large proportion of that development cost. Right now, only about 10% of compounds that enter clinical development make it to the marketplace. It was estimated that the effective application of genomics technologies could reduce that staggering investment by as much as $300 million and two years. Many are optimistic that pharmacogenomics can play a major role in increasing drug development productivity. The real cost savings will be that we have fewer failures going into development. Some anticipate that pharmacogenomics will contribute to all pharmaceutical products, from new candidates to old warhorses. Many pharmaceutical companies are already using pharmacogenomics to screen participants in clinical trials. The FDA appears to be solidly on board the pharmacogenomics bandwagon as well. The agency has reportedly been working hard recently to acquire the necessary inhouse expertise to facilitate the submission, interpretation, and implementation of pharmacogenomics data.

26.14 Applied Pharmacogenetics Pharmacogenomics will undoubtedly be used to improve future health care and clinical research in different ways (Roden et al., 2006). Whereas treatment allocation has been based mainly on phenotype, genetic characterization will help researchers to identify suitable subjects for clinical trials, to facilitate interpretation of the results of clinical trials, and to identify novel targets for future drugs or new markets for current products. As interindividual variability in drug response is a substantial clinical problem, the second major objective of pharmacogenomic research is to decrease adverse responses to therapy through determination of adequate therapeutic targets and genetic polymorphisms that alter drug specificity and toxicity. Most of the promise of pharmacogenomics remains to be fulfilled. However, the concept of using known genetic associations to prevent patients from taking drugs that would likely be ineffective or harmful is already available and used in clinical practice in certain specific arenas (Phillips et al., 2001), thanks mainly to the steady progress made in pharmacogenetics over the past several decades (Cariou et al., 2002). Cancer therapy today includes two shining examples of applied pharmacogenetics. First, there is now a commercially available diagnostic test measuring a

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patient’s ability to produce the metabolic enzyme thiopurine S-methyltransferase (TPMT), which is essential for the metabolism of thiopurine medications used to treat acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Genetic testing gives clinicians the ability to classify ALL patients according to their TPMT genotype, which allows optimized dosing. Doses in patients with alleles rendering them deficient in TPMT (who are thus less tolerant of thiopurine medications) are reduced by as much as 95%. This means TPMT-deficient patients can tolerate the drug, yet enough is still metabolized to retain efficacy. Second, the breast cancer drug trastuzumab (trade name Herceptin), which is marketed in tandem with a diagnostic test, is often cited as an early indicator of the value of the concept. Trastuzumab is effective only sin the 25–30% of breast cancer patients whose tumors overexpress the human epidermal growth factor receptor (HER2) protein. The drug was developed specifically to exploit that characteristic; it binds to HER2, which slows tumor growth. The diagnostic test measures HER2 expression in the tumor and is thus predictive of the potential efficacy of the drug; patients who do not overexpress HER2 are not given the drug, because it will not work. This is a unique combination today. But such diagnostic-agent pairings will become more commonplace as pharmacogenomics progresses and strides are made in disease genetics, in which a variety of diseases (particularly cancer) are being genetically subclassified, often significantly redefining treatment strategies. An intermediate step toward such pairings is illustrated by work being done with the cytochrome P450 (CYP450) family of enzymes, which is responsible for a large segment of human drug metabolism. It is the metabolic pathway of choice for about 60% of the drugs on the market today. It has also been the focus of a great deal of research attention through the years, and the numerous CYP450 subtypes are well characterized, as are the important phenotypes of variation in response. Several companies now offer CYP450 genotyping tests to the pharmaceutical industry for clinical trial subject inclusion/exclusion based upon metabolic profile, and now such tests are making their way into the clinical diagnostic marketplace. Gentris, for example, soon expects to market five kits to physicians for pharmacogenetic testing of their patients. Genelex Corporation of Seattle, Washington, has taken the concept one step further, marketing tests directly to the public for three of the major CYP450 pathways–CYP2D6, CYP2C9, and CYP2C19. Once a consumer has placed an order for the test, Genelex sends them a blood collection kit, and the consumer either sees their own doctor or Genelex will refer them to a phlebotomist in their area.

26.15 Ethical Concerns On one level, the ethical issues involved with pharmacogenomics are similar to those raised by genomics.In general, broad concerns about research integrity, privacy, confidentiality, informed consent, the specter of genetic discrimination or stigmatization, and access to information or to specialized care (Moldrup, 2001) are some of the chief concerns.

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Molecular Diagnostic Methods for Optimizing Drug Therapy

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Many observers, however, feel that the ethical issues specific to pharmacogenomics are actually somewhat less problematic than the ethical lightning rods attached to genetics or even to pharmacogenetics. One crucial difference lies in the nature of the information itself, which typically applies to individual patients as opposed to larger groups. Society, industry, groups and individuals appreciate the prospect of pharmacogenomics very differently. Secondly, there is a lack of research into the post-marketing implications of pharmacogenomics. An extensive focus on the ethical, social and legal implications of pharmacogenomics, in terms of both pre- as well as post-marketing issues, is essential. Also, a multidisciplinary approach which includes individual and group opinions in an upfront manner in the research and development process is essential. Otherwise, there is a substantial risk that the positive prospects of pharmacogenomics will not survive due to fear and a lack of acceptance and understanding on the part of the general public. The ethical issues connected to pharmacogenomics are perhaps less urgent. The reason is that the core of pharmacogenomics is pharmaceutical intervention (Bellver, 2002). The fact that we’re talking about genomics that a ‘pharmaco-’ attached to it means that the purpose is treatment, not primarily diagnosis of the disease. Today, pharmacogenomics is still predominantly a research endeavor, and the nature of informed consent is presently the most prominent ethical consideration for investigators. Achieving the appropriate level of informed consent could prove to be a major challenge, as participants will need to comprehend that their DNA could be used for multiple experiments over a long period of time, and that in many cases, their samples will not be anonymous. In many pharmacogenomics studies, anonymization of samples defeats the purpose of drawing associations between drug response and populations. On the other hand, participants’ access to the information discovered about their genomes is also a thorny issue for researchers. Obligations need to be negotiated in advance during the informed-consent process, and must be in place in situations when an individual is found to have a particular abnormality in his or her response to certain drugs, or perhaps an increased risk of disease due to a genetic susceptibility.

26.16 Molecular Diagnostic Methods for Optimizing Drug Therapy The potential is enormous for pharmacogenomics to yield a powerful set of molecular diagnostic methods that will become routine tools with which clinicians will select medications and drug doses for individual patients. A patient’s genotype needs to be determined only once for any given gene, because except for rare somatic mutations, it does not change. Genotyping methods are improving so rapidly that it will soon be simple to test for thousands of single-nucleotide polymorphisms in one assay. It may be possible to collect a single blood sample from a patient, submit a small aliquot for analysis of a panel of genotypes (e.g., 20,000

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single-nucleotide polymorphisms in 5000 genes), and test for those that are important determinants of drug disposition and effects. In our opinion, genotyping results will be of greatest clinical value if they are reported and interpreted according to the patient’s diagnosis and recommended treatment options (Evans and Relling, 1999).

26.17 Challenges for the Future There are a number of critical issues that must be considered as strategies are developed to elucidate the inherited determinants of drug effects. A formidable one is that the inherited component of the response to drugs is often polygenic. Approaches for elucidating polygenic determinants of drug response include the use of anonymous single-nucleotide polymorphism maps to perform genome-wide searches for polymorphisms associated with drug effects, and candidate-gene strategies based on existing knowledge of a medication’s mechanisms of action and pathways of metabolism and disposition. Both these strategies have potential value and limitations. However, the candidate-gene strategy has the advantage of focusing resources on a manageable number of genes and polymorphisms that are likely to be important, and it has produced encouraging results in a number of studies. The limitations of this approach are the incompleteness of knowledge of a medication’s pharmacokinetics and mechanisms of action. Gene-expression profiling and proteomic studies are evolving strategies for identifying genes that may influence drug response. Pharmacogenomics offers the hope of using the genetic profiling to personalized and thereby improve medical treatment of a variety of a diseases, including hypertension, depression, cancer, and HIV. One of the most important challenges in defining pharmacogenetic traits is the need for well-characterized patients who have been uniformly treated and systematically evaluated to make it possible to quantitate drug response objectively (Sadee, 2002, 1999). To this end, the norm should be to obtain genomic DNA from all patients enrolled in clinical drug trials, along with appropriate consent to permit pharmacogenetic studies. Because of marked population heterogeneity, a specific genotype may be important in determining the effects of a medication for one population or disease but not for another; therefore, pharmacogenomic relations must be validated for each therapeutic indication and in different racial and ethnic groups. Remaining cognizant of these caveats will help ensure accurate elucidation of genetic determinants of drug response and facilitate the translation of pharmacogenomics into widespread clinical practice. Much is needed before the potential benefits of pharmacogenomics can be fully realized. Identification and analysis of millions of SNPs that might influence the response to a specific drug or class of drugs is complex. Even after SNPs of interest have been catalogued, drug companies may limit drug development to agents targeting treatment of those comprising large groups. Development of drugs anticipated to be effective in only small cohorts may lie fallow. Improved DNA microarray and related technologies are needed to expedite SNP screening in a timely and cost effective manner. Extensive training of physicians will be required. Diverse ethical

References

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and legal issues including ownership of rights to genetic information, privacy, and insurance implications require resolution The outcome of drug therapy is often unpredictable, ranging from beneficial effects to lack of efficacy to serious adverse effects (Dan et al., 2006). How quickly pharmacogenomics develops into a useful clinical toolkit will depend greatly on how policymakers respond to policy issues associated with both genetic testing and targeted drug development, including concerns about safety, access, cost, and potential ethical and social implications. Overcoming these challenges holds the promise of improving new drug development and ultimately individualizing the selection of appropriate drugs and dosages for individual patients. Finally, pharmacogenomics is likely to transform the way clinical trials are conducted by allowing for the selection of a more homogeneous study population, thereby reducing the size and cost of clinical investigation.

References Bellver Capella, V., 2002, Ethics, politics, and rights in pharmacogenomics. Law Hum Genome Rev 17:31–55. Blake, C.A., Sobel, B.E., 2008, Progress in pharmacogenomics and its promise for medicine. Exp Biol Med 233:1482–1483. Cariou, A., Chiche, J.D., Charpentier, J., Dhainaut, J.F., Mira, J.P., 2002, The era of genomics: impact on sepsis clinical trial design. Crit Care Med 5:341–348. Dan, M., Roden, M.D., Russ, B., et al., 2006, Pharmacogenomics: challenges and Opportunities. Annals of Internal Medicine 145(10):749–757. Eichelbaum, M., Ingelman-Sundberg, M., Evans, W.E., 2006, Pharmacogenomics and individualized drug therapy. Annu Rev Med 57:119–137. Evans, W.E., Relling, M.V., 1999, Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286:487–491. Gupta, M., Kaur, H., Grover, S., Kukreti, R., 2008, Pharmacogenomics and treatment for dementia induced by Alzheimer’s disease. Pharmacogenomics 9:895–903. Johnson, J.A., 2001, Drug target pharmacogenomics: an overview. Am J Pharmacogenomics 1:271–281. Johnson, J.A., Evans, W.E., 2001, Pharmacogenomics: the inherited basis for inter-individual differences in drug response. Annu Rev Genomics Hum Genet 2:9–39. Johnson, J.A., Evans, W.E., 2002, Molecular diagnostics as a predictive tool: genetics of drug efficacy and toxicity. Trends Mol Med 8:300–305. Kirchheiner, J., Roots, I., Goldammer, M., Rosenkranz, B., Brockmöller, J., 2005, Effect of genetic polymorphisms in cytochrome p450 (CYP) 2C9 and CYP2C8 on the pharmacokinetics of oral antidiabetic drugs: clinical relevance. Clin Pharmacokinet 44:1209–1225. Kirchheiner, J., Seeringer, A., 2007, Clinical implications of pharmacogenetics of cytochrome P450 drug metabolizing enzymes. Biochim Biophys Acta 1770:489–494. Kirchheiner, J., Seeringer, A., Brockmöller, J., 2006, State of the art of pharmacogenetic diagnostics in drug therapy. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 49:995–1003. Krynetski, E.Y., Evans, W.E., 1998, Pharmacogenetics of cancer therapy: getting personal. Am J Hum Genet 63:11–16. Lazarou, J., Pomeranz, B.H., Corey, P.N., 1998, Incidence of adverse drug reactions in hospitalized patients. JAMA 279:1200–1205. Lee, S.S., 2009, Pharmacogenomics and the challenge of health disparities. Public Health Genomics 2:170–179.

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López-López, M., Guerrero-Camacho, J.L., Familiar-López, I.M., 2004, Pharmacogenomics: the quest for individualized therapy. Rev Neurol 39:1063–1071. Mancinelli, L., Cronin, M., Sadee, W., 2000, Pharmacogenomics: The promise of personalized medicine. AAPS Pharm Sci 2:4. Moldrup, C., 2001, Ethical, social and legal implications of pharmacogenomics: a critical review. Community Genet 4:204–214. Phillips, K.A., Veenstra, D.L., Oren, E., Lee, J.K., Sadee, W., 2001, The potential role of pharmacogenomics in reducing adverse drug reactions: A systematic review. JAMA 286:2270–2279. Phillips, K.A., Veenstra, D., Sadee, W., 2000, Implications of the genetic revolution for health services research: Pharmacogenomics and improvements in drug therapy. Health Serv Res 35:128–140. Roden, D.M., Altman, R.B., Benowitz, N.L., Flockhart, D.A., Giacomini, K.M., Johnson, J.A., Krauss, R.M., McLeod, H.L., Ratain, M.J., Relling, M.V., Ring, H.Z., Shuldiner, A.R., Weinshilboum, R.M., Weiss, S.T. Pharmacogenetics Research Network, 2006, Pharmacogenomics: challenges and opportunities. Ann Intern Med 145:749–757. Sadee, W., 1999, Pharmacogenomics. Brit Med J 319:1286. Sadee, W., 2002, Pharmacogenomics: The Implementation Phase. AAPS Pharm Sci 4:611–614. Shai, R.M., Reichardt, J.K., Chen, T.C., 2008, Pharmacogenomics of brain cancer and personalized medicine in malignant gliomas. Future Oncol 4:525–534. Shi, J., Chen, S., 2002, Pharmacogenomics genomics approaches to optimizing drug therapy. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 19:156–158. Terra, S.G., Johnson, J.A., 2002, Pharmacogenetics, pharmacogenomics, and cardiovascular therapeutics: the way forward. Am J Cardiovasc Drugs 2:287–296. Tolle, R., 2001, Information technology tools for efficient SNP studies. Am J Pharmacogenomics 1:303–314.

Chapter 27

Gene Technology in Forensic Sciences

Abstract Recombinant DNA technology can provide novel and powerful methods for forensic science application. Human genomic DNA can be analyzed directly for individual identification and paternity testing on the basis of polymorphism in its sequence. Restriction fragment length polymorphism (RFLP) testing, STR (microsatellite) and mitochondrial DNA analysis (mtDNA) is suitable for examination of the forensic biological samples (bloodstains, hairs, seminal stains, bones, tooth). Using a combination of single locus probe (SLP) that varies highly among individuals, a DNA fingerprint or profile can be made. Mitochondrial DNA RFLPs may also suggest the characteristics of the human races. Keywords Forensic · Genetic markers · DNA typing · RFLP · STR · Gene technology · DNA profiling · RFLP · STR · Fingerprint · PCR based methods · Polymorphism · Amplification

27.1 Prologue Forensic science has witnessed dramatic changes in the field of human identification. DNA can be used to identify the perpetrator of a murder (Linacre and Graham, 2002). The forensic potential of DNA profiling for human identification burst upon the scene in late 1985. Since then, forensic DNA testing has gone through several phases of technological and operational advance resulting in more rapid analysis, extension of testing to ever smaller biological samples, and a de facto international standardization based on use of commercial kits. The technology has altered by adopting novel methods developed originally for use in the field of medical genetics. There can be few areas of endeavour in molecular genetics that have captured the imagination and interest of the public as much as DNA typing. DNA typing is renowned as a technique that is used in forensic investigations to match criminals against samples obtained from crime scenes, to identify individuals from their remains and to determine paternity. It is also an excellent example of a new technology based on sound scientific principles and on the successful application of those principles to a commercial field. M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_27,

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27.2 Concept Like many applications of molecular diagnostics, the field of forensic biology is undergoing a phase of expansion and diversification. Forensic science is the application of a broad spectrum of sciences to answer questions of interest to the legal system. Among the branches of Forensic sciences, criminalistics and Forensic DNA analysis rely on the gene technology using DNA profiling technique. Criminalistics is the application of various sciences to answer questions relating to examination and comparison of biological evidence, trace evidence, impression evidence (such as fingerprints, footwear impressions, and tyre tracks), controlled substances, ballistics, firearm and toolmark examination, and other evidence in criminal investigations. Typically, evidence is processed in a crime lab. Forensic DNA analysis takes advantage of the uniqueness of an individual’s DNA to answer forensic questions such as determining paternity/maternity or placing a suspect at a crime scene. In the last few years, DNA typing procedures have become increasingly important in the fields of forensic science and forensic medicine. The growth of forensic DNA databases and adoption of sophisticated analytical methods have catalyzed this increasing role. The range of molecular markers exploited in the fight against crime is beginning to increase too, and genes implying personal or physical characteristics are emerging in the research literature. DNA profiling has greatly changed the way in which human identification is performed for the purpose of a forensic investigation. DNA typing is probably one of the most important advances in the forensic sciences in recent years. While most DNA is the same from one person to another, several sections can show marked variability in the sequence of the monomers. The DNA sequence of a person is therefore individual and can be shown to be theirs beyond reasonable doubt. The first application of DNA typing to forensic science was based on restriction fragment length polymorphisms (RFLP). More recent methods of DNA typing are based on the technique of polymerase chain reaction (PCR). This allows the amplification of minute amounts of DNA (as small as 1 ng of material) giving higher sensitivity for DNA typing. DNA typing is now possible from samples as small as a few cells on the end of a hair that has been pulled out. Genetic characterization of individuals at the DNA level enables identity testing from a minimal amount of biological specimen in cases of sexual assault, homicide, and unknown human remains. Also paternity testing is changing from level of gene products to the genomic level (Sajantila and Budowle, 1991). Currently, millions of samples from blood, semen, hair and tissues are analyzed to determine the origin of the samples.

27.3 DNA Typing and Genetic Markers Since the introduction of DNA polymorphism analysis techniques to forensic science, forensic identification research has made radical, astonishing progress at a rate that has already rendered the initial methodologies obsolete. The ability to extract

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and type DNA from forensic samples has revolutionized the field of forensic serology. DNA extraction now can be quickly and efficiently performed by various kinds of commercially available kits. DNA can also be purified by the potassium iodine method (Shiono, 1996) or phenol extraction method. The analysis of DNA isolated from forensic biological evidence provides valuable information relating to the identification of the source of the sample (Misawa, 1994). The advent of PCR has enabled the use of relatively crude and minute DNA as amplification templates while many kinds of new detection methods for analyzing the amplified products have also been developed. Organism can be identified by examination of DNA sequences unique to that species. In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions called genetic markers. To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person and use the data to create a DNA profile of that individual (sometimes called a DNA fingerprint). There is an extremely small chance that another person has the same DNA profile for a particular set of regions (Fig. 27.1) Scientists find the markers in a DNA sample by designing small pieces of DNA or probes that will each seek out and bind to a complementary DNA sequence in the sample. A series of probes bound to a DNA sample creates a distinctive pattern for an individual. Forensic scientists compare these DNA profiles to determine whether the suspect’s sample matches the evidence sample. A marker by itself usually is not unique to an individual; if, however, two DNA samples are alike at four or five regions, odds are great that the samples are from the same person. If the sample profiles don’t match, the person did not contribute the DNA at the crime scene. If the patterns match, the suspect may have contributed the evidence sample. While there is a chance that someone else has the same DNA profile for a particular probe set, the odds are exceedingly slim. Experts point out that using DNA forensic technology is far superior to eyewitness accounts, where the odds for correct identification are about 50:50. The more probes used in DNA analysis, the greater the odds for a unique pattern and against a coincidental match, but each additional probe adds greatly to the time and expense of testing. Four to six probes are recommended to detect polymorphism. Since the introduction of DNA polymorphism analysis techniques to forensic science, forensic identification research has made radical, astonishing progress at a rate that has already rendered the initial methodologies introduced fifteen years ago obsolete (Fukushima, 1999). Although many minisatellites such as MCT118, YNZ22, COL2A1, and ApoB were highlighted at the beginning of 1980s, none of these loci, with the exception of MCT118, have proved useful for forensic DNA application due to their low amplification efficiency. On the other hand, STR loci containing four base pair repeat sequences have been used routinely for human identification since the mid-1990s. In the near future, the highly efficient STR should be selected as a consensus core marker in Japan. STR systems located on the Y chromosome are widely used in forensic science for the identification of male individuals. These systems have a special significance in forensic science cases where mixtures of male and female DNA are analyzed, as happens in cases of rape or

458 Fig 27.1 Method of DNA fingerprinting

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other sexual crimes. The characteristics of high copy number, maternal inheritance, and high degree of sequence variability make mtDNA a powerful tool for forensic identification. Most of the variations in mtDNA among individuals are found within the displacement loop (D-loop). In all population groups, mtDNA sequences can be useful for discriminating among unrelated individuals. Now it is necessary to get as much as possible individual genetic information as quickly as possible in order to enable individual identification.

27.4 DNA Based Methods for Identification of Individuals Previously, genetic marker typing was limited to the analysis of blood group markers (Sasaki and Shiono, 1996) and soluble polymorphic protein markers. Because the number of suitable markers expressed in particular fluids and tissues is relatively small, and because mixtures of fluids cannot be separated for conventional genetic marker typing, a suspect frequently cannot be included or excluded as a fluid donor in a case. However, the development of methods to extract DNA from virtually all biological specimens has greatly expanded the potential for individual identification. Of particular importance was the ability to extract mixtures of sperm cells and epithelial cells found in sexual assault cases such that the DNA from the sperm cells could be typed independently of the DNA from the victim’s epithelial cells.

27.4.1 Non-PCR Based Methods Restriction fragment length polymorphism (RFLP) analysis was the first DNAbased method applied to problems of individual identification (Reynolds et al., 1999). It can arise when an individual may have an additional recognition site for a particular restriction endonuclease in a given area of the genome or, an individual may be missing a recognition site for a particular restriction endonuclease in a given area of the genome. This method, while powerful in its ability to differentiate individuals, is limited by the quantity and quality of DNA required for an unambiguous result and by the amount of time it takes to obtain a result. Despite these limitations, several laboratories are using RFLP analysis successfully for the detection of polymorphisms in forensic DNA case samples. Although either of the above situations can lead to polymorphic differences in the population, many of the specific loci that are used in criminal forensic cases and in paternity cases involve repetitions of a core sequence at a given genetic locus. The length of each repeat unit and the number of repeat units varies. Therefore, the length of the fragment generated by a restriction enzyme that cuts on either side of (but not within) the locus also varies. These types of loci are referred to as Variable Number of Tandem Repeats loci (VNTRs).

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27.4.2 PCR Based Methods While the field of forensic serology was being revolutionized by the prospect of DNA analysis, the field of molecular biology was being revolutionized by the invention of the polymerase chain reaction (PCR), which ultimately has an impact on every area of biological science (Lee et al., 1994). The PCR DNA amplification technology is ideally suited for the analysis of forensic DNA samples in that it is sensitive and rapid and not as limited by the quality of DNA as the RFLP method. a. Ampli Type PM & DQA1 – Sequence Polymorphisms. The Commercial availability of the AmpliType PM & DQA1 system from Applied Biosystems, INC (ABI; formerly Perkin-Elmer) was the first PCR based DNA Typing systems. The loci involved in this system are all sequence polymorphisms that are detected/ delineated by hybridization to allele-specific oligonucleotide (ASO) probes. The discriminatory power of this system is much more limited than RFLP analysis (approximately 1:2000) because only a limited number of alleles (and therefore, a limited number of genotypes) exist in the population. Nevertheless, the system is useful when evidence samples yield limited amounts of DNA and/or DNA that is too degraded for RFLP analysis. The AmpliType PM & DQA1 system tests for polymorphisms at 6 loci namely, low Density Lipoprotein Receptor (LDLR), Glycophorin A (GLYPA), Hemoglobin G Gamma Globulin (HBGG), D7S8, Group-Specific Component (GC), HLADQA1 (Comey et al., 1993). The basic steps in the procedure are: 1. Simultaneous PCR amplification of the six loci using primers that have been conjugated to biotin. 2. Hybridization (allele-specific) of the PCR products using a reverse dot-blot method in which the ASO probes have been impregnated onto membrane filter strips in specific locations; two strips are used: one for the DQA1 locus and one for the remaining 5 loci of the Polymarker (PM). 3. Binding of a horseradish peroxidase/streptavidin conjugate to the hybridized biotinylated PCR product. 4. Colorimetric detection of the hybridized PCR product-biotin/HRP-streptavidin complex using a chromogenic substrate for the HRP. Because of its low Power of Discrimination (Pd) this system is seldom used anymore. However, it is still commercially available and used occasionally. b. D1S80 – Amplified Fragment Length Polymorphism (AFLP). VNTRs, such as described above for RFLP analysis, can also be detected by Polymerase Chain Reaction (PCR) Amplification. Primers are designed to anneal to sequences in the DNA that flank the VNTR. PCR Amplification of the material between the primer locations includes the VNTR region. Therefore, any allelic differences between individuals will be evidenced by different lengths for the amplification product among individuals tested. The D1S80 locus has been used this way. It has a core repeat unit of 16 bp, which is repeated 14–41 times.

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The basic process involves: 1. Processing appropriate samples to obtain the DNA. 2. PCR Amplification of a region of the DNA containing a polymorphic VNTR (i.e. D1S80). 3. Separating the resulting DNA amplification products by electrophoresis. 4. Analysis of the resulting pattern by UV transillumination of Ethidium bromide stained gels or by Silver Staining to determine inclusion or exclusion. c. Short Tandem Repeats (STRs; Microsatellites). Short Tandem Repeat sequences (STRs) are similar to VNTRs in that they involve tandem repeats of a core sequence in variable numbers among the population to produce a polymorphic distribution. The major difference is that the core sequence is usually only 3 or 4 nucleotides in length (VNTR core sequences can be 16 or more nucleotides). Usually a tighter range of allele’s results. The small size of the STRs used in forensic DNA profiling (amplimers range from 100 to 500 bp) allows for more efficient amplification by PCR and also allows the use of DNA that has been degraded more significantly because even small pieces of DNA may contain intact STR sites. Primers are designed to anneal to sequences in the DNA that flank the STR. PCR Amplification of the material between the primer locations includes the STR region. Therefore, any allelic differences between individuals will be evidenced by different lengths for the amplification product among individuals tested (Divne and Allen, 2005). d. Multiplex STRs. A number of STR loci have been identified with nonoverlapping allelic distributions. This allows the use of multiplex PCR amplification and separation of multiple loci in a single lane of a polyacrylamide gel. The use of different fluorescent dyes allows the multiplex amplification of STR loci that have overlapping allele ranges as long as a separate dye is used for each overlapping STR. The dyes are coupled to the primers used for the amplification so that the different loci can be readily identified. e. Mitochondrial D-Loop DNA (a sequence polymorphism). Recently, it has been recognized that information in the mitochondrial DNA (mtDNA) coding region can provide additional forensic discrimination with respect to the standard typing of the D-loop region, increasing the forensic power of mtDNA testing, which is sometimes rather limited(Wu et al., 2008). The characteristics of high copy number, maternal inheritance, and high degree of sequence variability make mtDNA a powerful tool for forensic identification (Fukushima, 1999). Two particular sequences within this region tend to mutate with extremely high frequency and thus allow for highly variable sequence polymorphisms among unrelated individuals. The PCR method using mitochondrial DNA analysis (mtDNA) is suitable for examination of the forensic biological samples (bloodstains, hairs, seminal stains, bones, tooth). It is also useful for tracking new variations arising by mutation. They are present in 100s–1000s of copies per cell. Due to their small size and circular form, it is resistant to degradation. So they can also be found in old samples (e.g. bones that are found years after the crime was committed), in dead cells such as hair shafts, bones and

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teeth. Mitochondrial DNA RFLPs and 9-bp deletion type of mtDNA may suggest the characteristics of the human races (Misawa, 1994) f. Y-Chromosome STRs. The Y chromosome is inherited in a direct patrilinear manner. Since it is only found in males it can be useful in a variety of situations where a mixture of male and female donors could obscure the results. For sex identification of bloodstains, bleached skeletons and teeth, Southern blot hybridization with Y-chromosome specific probe (pHY10; 3.4 kb) can be an appropriate method (Misawa, 1994). This method is also used for tracking patrilineal descent, hemizygous individuals. It can also be used on mixed samples regardless of the relative proportions of male and female material (vaginal smears; fingernail scrapings; fingernail clippings, etc.). g. SNP. The range of molecular markers exploited in the fight against crime is beginning to increase too, and genes implying personal or physical characteristics are emerging in the research literature. Of a number of DNA marker typing techniques for personal identification in the field of forensic medicine, polymorphic short tandem repeat (STR) typing is currently the most frequently used technique. However, the multiplex STR method is time consuming. In contrast, single nucleotide polymorphism (SNP) detection methods are relatively rapid and amenable to high throughput (Hu and Wu, 2001). Single nucleotide polymorphisms (SNPs) are emerging as new markers of interest to the forensic community because of their abundance in the human genome, their low mutation rate, the opportunity they present of analyzing smaller fragments of deoxyribonucleic acid (DNA) than with short tandem repeats. This method is also important in degraded DNA samples and the possibility of automating the analysis with high-throughput technologies (Sobrino and Carracedo, 2005). The discrimination power of each SNP is inferior to that of an STR, but a combination of many SNPs could realize a high discriminating power (Hiratsuka et al., 2005). These have a particular advantage in the analysis of degraded or poor samples, which are often all that is available in forensics (Petkovski et al., 2005). h. DNA Forensics Databases. Like many applications of molecular diagnostics, the field of forensic biology is undergoing a phase of expansion and diversification. The growth of forensic DNA databases and adoption of sophisticated analytical methods have catalyzed this increasing role (Walsh, 2004). The Combined DNA Index System, CODIS, the national DNA databank, blends computer and DNA technologies into a tool for fighting violent crime. The current version of CODIS uses two indexes to generate investigative leads in crimes where biological evidence is recovered from the crime scene. The Convicted Offender Index contains DNA profiles of individuals convicted of felony sex offenses (and other violent crimes). The Forensic Index contains DNA profiles developed from crime scene evidence. All DNA profiles stored in CODIS are generated using STR (short tandem repeat) analysis. CODIS utilize computer software to automatically search its two indexes for matching DNA profiles. Law enforcement agencies at federal, state, and local levels take DNA from biological evidence (e.g., blood and saliva) gathered in crimes that

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have no suspect and compare it to the DNA in the profiles stored in the CODIS systems. If a match is made between a sample and a stored profile, CODIS can identify the perpetrator. This technology is authorized by the DNA Identification Act of 1994. As more offender DNA samples are collected and law enforcement officers become better trained and equipped to collect DNA samples at crime scenes, the backlog of samples awaiting testing throughout the criminal justice system is increasing dramatically.

27.5 Ethical, Legal, and Social Concerns About DNA Databanking The primary concern is privacy. DNA profiles are different from fingerprints, which are useful only for identification. DNA can provide insights into many intimate aspects of people and their families including susceptibility to particular diseases, legitimacy of birth, and perhaps predispositions to certain behaviors and sexual orientation. This information increases the potential for genetic discrimination by government, insurers, employers, schools, banks, and others. Collected samples are stored, and many state laws do not require the destruction of a DNA record or sample after a conviction has been overturned. So there is a chance that a person’s entire genome may be available regardless of whether they were convicted or not. Although the DNA used is considered “junk DNA”, single tandem repeated DNA bases (STRs), which are not known to code for proteins, in the future this information may be found to reveal personal information such as susceptibilities to disease and certain behaviors. Practicality is a concern for DNA sampling and storage. An enormous backlog of over half a million DNA samples waits to be entered into the CODIS system. The statute of limitations has expired in many cases in which the evidence would have been useful for conviction.

27.6 Future of DNA Typing Systems The major problem for the foreseeable future is not increasing the Power of Discrimination but rather increasing throughput so that more samples can be processed in a reasonable period of time to ensure speedy trials. New instruments and new methodologies are being developed with this goal in mind. Adoption of new methodologies in the forensic community can be slow because of the need for extensive validation and the development of QA/QC protocols. • Capillary Array Electrophoresis – multiple capillary electrophoresis runs simultaneously (e.g. ABI PRISM 3700 runs 96 samples simultaneously)

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• MALDI-TOF Mass Spectrometry (Matrix Assisted Laser Desorption-IonizationTime of Flight) – analysis times on the order of seconds • STR Determination by Hybridization Arrays – silicon microchip hybridization – size sensitive results without electrophoretic separation • SNP Determination by Hybridization Arrays and other techniques for SNP detection • Laboratory automation – liquid handling robots • Computerized sample tracking programs • Computerized STR Interpretation programs • Portable systems that can be used at the crime scene – probably microchip systems. However, the operational context of forensic biology is unlike many other fields of science. Harmonizing technological breakthroughs with the requirements of law enforcement agencies and the complexities of the legal system is an added challenge and one which evokes ongoing debate. A new era in which forensic identification can be performed using microarray technology can be visualized in future.

References Comey, C.T., Budowle, B., Adams, D.E., Baumstark, A.L., Lindsey, J.A., Presley, L.A., 1993, PCR amplification and typing of the HLA DQ alpha gene in forensic samples. J Forensic Sci 38:239–249. Divne, A.M., Allen, M., 2005, A DNA microarray system for forensic SNP analysis. Forensic Sci Int 154:111–121. Fukushima, H., 1999, Forensic DNA analysis – past and future. Nihon Hoigaku Zasshi 53: 276–284. Hiratsuka, M., Tsukamoto, N., Konno, Y., Nata, M., Hashiyada, M., Funayama, M., Mizugaki, M., 2005, Forensic assessment of 16 single nucleotide polymorphisms analyzed by hybridization probe assay. Tohoku J Exp Med 207:255–261. Hu, R., Wu, X.Y., 2001, The single nucleotide polymorphisms and its application to forensic medicine. Fa Yi Xue Za Zhi 17:249–251. Lee, H.C., Ladd, C., Bourke, M.T., Pagliaro, E., Tirnady, F., 1994, DNA typing in forensic science. I. Theory and background. Am J Forensic Med Pathol 15:269–282. Linacre, A., Graham, D., 2002, Role of molecular diagnostics in forensic science. Expert Rev Mol Diagn 2:346–353. Misawa, S., 1994, Application of DNA polymorphism to forensic medicine. Rinsho Byori 42: 636–642. Petkovski, E., Keyser-Tracqui, C., Hienne, R., Ludes, B., 2005, SNPs and MALDI-TOF MS: tools for DNA typing in forensic paternity testing and anthropology. J Forensic Sci 50:535–541. Reynolds, R., Sensabaugh, G., Blake, E., 1999, Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction. Anal Chem 63:2–15. Sajantila, A., Budowle, B., 1991, Identification of individuals with DNA testing. Ann Med 23: 637–642. Sasaki, M., Shiono, H., 1996, ABO genotyping of suspects from sperm DNA isolated from postcoital samples in sex crimes. J Forensic Sci 41:275–278. Shiono, H., 1996, Personal identification using DNA polymorphism–the identification of forensic biological materials. Nihon Hoigaku Zasshi 50:320–330.

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Sorbrino, B., Carracedo, A., 2005, SNP typing in forensic genetics: a review. Methods Mol Biol 297:107–126. Walsh, S.J., 2004, Recent advances in forensic genetics. Expert Rev Mol Diagn 4:31–40. Wu, J., Ran, P., Zhang, B., Li, Y., Yan, J., Liao, M., Hou, Y., Zhang, L., 2008, Multiplex mutagenically separated polymerase chain reaction assay for rapid detection of human mitochondrial DNA variations in coding area. Croat Med J 49:32–38.

Chapter 28

Food Diagnostics

Abstract Food diagnostics is a relatively new and emerging area fuelled in large part by the ever-increasing demand for food safety. As a derivative of the larger medical diagnostic industry and other diagnostic areas (e.g., veterinary, environmental, and agricultural), the technology of food diagnostics is rapidly changing. Therefore, this field of application is used to outline principles, challenges, and current developments of DNA analysis in foods. In addition, DNA-based approaches are also essential in the areas of food authentication, detection of food-borne pathogenic micro organisms, and screening for food allergens. Keywords Food diagnostics · Food allergens · PCR · ELISA · Food safety · Food borne disease · Microbiology · Antibody · Immunomagnetic separation · Amplification · Sensor technology · Microarray

28.1 Prologue Today’s era is driving the development and commercialization of biotechnological products and processes for agriculture and food system. These processes and products have a major impact on maintaining and improving food safety. The ability of commercial food industry to identify food pathogens and unsafe residues resulted in an almost fivefold increase in food as recalled by major manufacturers since 1988. New technology allows Government regulatory agencies to identify a bacterial pathogen and trace it back to its source more rapidly. The key to this new technology is the availability of rapid food-safety diagnostics. These rapid food-safety diagnostics provide a quick answer before the food product enters the distribution system. For example, rapid screening for Salmonella typhimurium requires using PCR method is approximately 24 h, in contrast to 4–5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods (Malorny et al., 2004).


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Food Diagnostics

28.2 Concept Food Diagnostics is an emerging field which applies “modern” methods of detection of bacteria, viruses, parasites, chemicals, biotoxins, heavy metals and prions in all steps of the food chain from raw materials to end products. Using the molecular diagnostic techniques, the detection of a fragment of genetic material (nucleic acids, i.e., DNA or RNA) that is unique to the target pathogenic organism can be successfully accomplished. One of the most practical and useful applications of molecular tools is their specificity as they target genetic regions unique to the organism, and depending on the gene target, they can also yield valuable information about virulence properties of the organism. They are also invaluable in detecting and identifying infectious agents. In the last decade the need for methods to detect and to quantify DNA from genetically modified organisms (GMOs) has been a major driver for the development and optimization of PCR-based techniques. Throughout the world, food production, preparation and distribution have become increasingly complex, and raw materials are often sourced globally. Changes in food processing techniques, food distribution and the emergence of new food pathogens have changed the epidemiology of food-borne diseases. Foodborne microorganisms are continuously changing due to their inherent ability to evolve and their amazing capacity to adapt to different forms of stress. New primary production technologies and food manufacturing practices are introduced all the time; food consumption patterns and the demographic structure of many countries continue to change. The implementation of Food Safety should be seen as an ongoing process, which is influenced by environmental, socio-economical, political and cultural factors. Food safety issues need to be managed on a continuous basis, from a regional, national, and global point of view. New, flexible tools are required for evaluating and managing new food safety challenges. To guarantee the safety of foodstuffs producers have therefore shifted their focus towards the use of food safety management tools, most important HACCP (Hazard Analysis Critical Control Point), and the consequent application of hygienic measures, based on Good Manufacturing/Hygienic practice (GMP/GHP). Food safety management tools use input of scientific information to identify critical contamination points in the food chain and the production process, and design measures to control them. However, the lack of reliable data is often limiting the usefulness of this approach and therefore data collection is one of the priorities for future food safety strategies. In the absence of relevant data, other strategies might still have to be used to control food hazards. The Food and Drug Administration (FDA) has been involved in the regulation of in vitro diagnostic devices (IVDs or laboratory tests) since the introduction of the Medical Device Amendments of 1976. IVDs developed as kits or systems intended for use in multiple laboratories require review by the FDA before being marketed to ensure appropriate performance and labelling (Gutman, 1999).

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28.3 Tools of Molecular Diagnostics to Assess Food Quality Food safety concerns are best met with an eye toward all aspects of production. Our growing line of products for on-site testing assures comprehensive food safety throughout the processing chain. It must assure food safety, meet regulatory requirements and measure up to customer expectations. Certain company specializes in developing immunoassay tests for the food industry. This technology provides users with fast, accurate, easy-to-use tests that are cost effective, require little space and minimal capital investment. Whether it’s the rapid analysis of GMOs in crops, mycotoxins in grains or food pathogens in meat, dairy (Chotár et al., 2006) or processed foods. Molecular diagnostics help to reduce risks, protect brands and gain confidence in the safety of operations (Lauri and Mariani, 2008). The modern diagnostic techniques used to assess food diagnostics are as follows: (1) The application of ultra sounds to monitor the quality of food products without affecting their integrity, i.e., non-destructive or non-invasive practices; (2) The use of biosensors and electronic noses for evaluating the safety and quality of agricultural and horticultural products; (3) The immunoassays for detection of food toxins, pesticides, food allergens ect. (4) The nucleic acid based assays to characterize foodborne pathogens and to identify genetically-modified food or food components; (5) Protein-based technology to detect and identify genetically-modified food or food components, and (6) The detection of biochemical markers with rapid chromatographic techniques. Recent advances in molecular cloning and recombinant DNA techniques have revolutionized the detection of pathogens in foods. Traditional methods of identification of food-borne pathogens, which cause disease in humans, are timeconsuming and laborious, so there is a need for the development of innovative methods for the rapid identification of food-borne pathogens (Naravaneni and Jamil, 2005). Nontyphoidal Salmonella are highly prevalent food-borne pathogens. High-throughput sequencing of Salmonella genomes is expanding our knowledge of the evolution of serovars and epidemic isolates (Andrews-Polymenis et al., 2009). Genome sequences have also allowed the creation of complete microarrays. Microarrays have improved the throughput of in vivo expression technology (IVET) used to uncover promoters active during infection. In another method, signature tagged mutagenesis (STM); pools of mutants are subjected to selection. Changes in the population are monitored on a microarray, revealing genes under selection. Techniques such as in vivo expression technology (IVET) and signature-tagged mutagenesis (STM) provide alternatives for studying bacterial gene expression and growth requirements within these settings (Dudley, 2008).

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28.4 Food Allergy Over the past 2 decades, the prevalence of food allergy has been doubled. Major food allergens have been identified for many common foods (Nowack-Wegrzyn and Sampson, 2006). Clinically these immediate-type food-induced hypersensitivity reactions are characterised by a variety of signs and symptoms that occur within minutes or hours after consumption of the offending food. Reactions may be limited or more generalised with involvement of the skin, nose, eyes, and/or lungs. In more severe cases, cardiovascular symptoms including hypotension, shock, cardiac dysrhythmias and death can occur (Ballmer and Vieths, 2008). In food-allergic individuals, IgE is produced against naturally occurring food components, primarily glycoproteins that usually retain their allergenicity after heating and/or proteolysis. While adults tend to be allergic to fish, crustaceans, peanuts and tree nuts, children tend to be allergic to cow’s milk, egg white, wheat and soya products more frequently. “Emerging” food allergens include tropical fruits, sesame seeds, psyllium, spices and condiments. These allergies frequently represent a cross-allergy to an allergen derived from another source, e.g. pollens or natural rubber latex. Food allergy encompasses a variety of immune-mediated adverse reactions to foods (Ebo and Stevens, 2001). IgE-mediated, cell-mediated, and mixed-mechanism food allergy disorders are recognized. The diagnostic work-up of suspected food allergy includes the skin prick test (SPT), the measurement of food specific immunoglobulin E (IgE) antibodies using serologic assays, and more recently the atopy patch test (Niggemann et al., 2005). Laboratory diagnosis of food allergy relies heavily on the detection of food-specific IgE antibodies, but novel approaches include tests for T-cell-mediated disorders and tests for prediction of tolerance. OFC remains the diagnostic standard for food allergy (Lidholm et al., 2006). Management of food allergy now-a-days focuses on avoidance of the offending foods, nutritional support, and prompt recognition and treatment of acute food allergic reactions. Anti-IgE monoclonal antibody is the first potential therapy for food allergy that is under-going testing in clinical trials.

28.5 Food-Borne Disease Through various food items, many high-risk pathogens that cause disease in humans are transmitted (Nugen and Baeumner, 2008). Food-borne disease across the world costs billions of dollars annually. Approximately 30% of these diseases are caused by bacteria and their related toxic products (Mead et al., 1999). The bacteria species that are most frequently responsible for food contaminations are Salmonella typhimurium (meat, milk, and eggs), Shiga toxin-producing Escherichia coli O157:H7 (meat), Staphylococcus aureus (milk, cream, and meat), Clostridium perfringens (sausages, preserved food), Campylobacter jejuni (poultry, eggs), Vibrio parahaemolyticus (shellfish), Yersinia enterocolitica (meat, milk), and Listeria monocytogenes (dairy products) (Magliulo et al., 2006; Lukinmaa et al., 2004). Infections due to Yersinia, Shigella, Listeria, Campylobacter, Escherichia

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Commercial Rapid Food-Safety Diagnostics

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coli O157:H7 and Salmonella have recently reported to decrease dramatically, while infections due to Vibrio have increased. A more recent report indicated similar findings, with a decrease in Yersinia, Shigella, Listeria and Campylobacter cases, and again a significant increase in Vibrio infections. The declining rates of infection due to Listeria monocytogenes and E. coli O157:H7 are likely a result of increased awareness. The FDA, USDA and EU have all implemented a zero-tolerance rule for L. monocytogenes in ready-to-eat (RTE) foods. Similarly, the USDA’s Food Safety and Inspection Service (USDA-FSIS) has declared E. coli O157:H7 in raw ground beef to be an adulterant and therefore unfit for human consumption. Food safety practices have vastly improved in the processing environment as a result of these regulatory actions, as evidenced by the decreasing rates of infection by both Listeria and E. coli O157:H7. However, of the 121 foodborne outbreaks reported through FoodNet in 2005, almost half (49%) of the reported cases were attributed to viruses, and the number of Vibrio infections is on the rise (Nugen and Baeumner, 2008). Therefore, while the “hot” organism may change and while food safety practices are improving, there remains a growing need for enhanced means of food pathogen detection. The majority of food-borne disease outbreaks result from unintentional contamination of a product as a result of inappropriate processing or handling. Intentional contamination of our food supply with biological or chemical agents also is a significant threat. Bioterrorist attacks on our food supply could be covert or announced and could be accomplished with selected bacterial pathogens or toxins. A key step in establishing an effective food-safety program in a forward-deployed theater is to have adequate laboratory diagnostics that can identify and characterize rapidly any agent that could cause a food-borne disease outbreak. To enhance current food-safety and -quality programs, the commercial rapid-screening food laboratory diagnostics are used. Some capabilities of these diagnostics include: • • • • •

Microbiology testing of both liquid and solid foods. Food surface sanitation validation. Dairy product shelf-life prediction testing. Pesticide analysis of fresh fruits and vegetables. Continuous internal product temperature monitoring of perishable food while in transit.

Commercial rapid food-safety diagnostics have been procured and deployed throughout the whole world as an integral part of active food-safety surveillance program.

28.6 Commercial Rapid Food-Safety Diagnostics Today’s consumers demand safe food. The commercial food industry’s ability to identify bacterial pathogens and unsafe residues has resulted in an almost fivefold increase in food recalls by major manufacturers since 1988. New technology allows Government regulatory agencies to identify a bacterial pathogen and trace it back to

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its source more rapidly. The key to this new technology is the availability of rapid food-safety diagnostics. These rapid food-safety diagnostics provide a quick, “positive or negative” answer before the food product enters the distribution system. Tests take up to 30 h to complete because of the requirement for a bacterial growth enrichment period. This growth enrichment period is necessary to increase the total number of bacteria so they can be detected using current technology. A negative answer means that the product does not contain that particular bacteria or toxin and no further testing is required. A positive answer means that further testing is needed at a reference laboratory using standard laboratory methods to confirm the actual presence of the bacteria or toxin. For example, rapid screening for Salmonella typhimurium requires approximately 24 h to complete.

28.7 Methods for Analysis of Food Food is a difficult test matrix since it is extremely varied in its chemical composition, contains many different ingredients, and often contains an intrinsic microbial flora with varying amounts of shelf-limiting bacteria and even pathogens. Furthermore, food is processed with varying technologies and stored under different conditions. During processing bacteria might be damaged sub lethally with subsequent problems of detection. As a result, different enrichment protocols might be necessary. Microbiological methods are divided into traditional or conventional methods (Cancrini and Iori, 2004) and rapid methods (Bennett et al., 1998). Culture-based methods used for microbial detection and identification are simple to use, relatively inexpensive, and sensitive. Traditional methods of identification of food-borne pathogens, which cause disease in humans, are time-consuming and laborious, so there is a need for the development of innovative methods for the rapid identification of food-borne pathogens. Conventional methods are still considered the gold standard and are often required by national and international regulatory agencies as official control methods routine microbiology laboratories. Similar to conventional tests, the first-generation in deoxyribonucleic acid assays determine only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or microarrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting. These Rapid Methods offer advantages in analysis time but also give the possibility to eliminate labour intensive steps and give the potential for automation. Due to the speed-up of the sampling process, sample treatment, detection/ enumeration procedure, the output of the laboratory can be improved(Fig. 28.1).

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Fig 28.1 Diagnostic tools and strategies implemented for food safety

28.7.1 Rapid Methods A new method must have high sensitivity, high specificity, high precision (repeatability), at the same time rapid, robust and cheap. There is currently no method that will fulfill all requirements. Antibody-based methods as represented by the ELISA assay and DNA based methods as the polymerase chain reaction are the most widely used technologies in food diagnostics today. Also immunomagnetic separation (IMS) is exploited in a number of commercially available kits and will become an even more important technology in the future. While some microarray based systems are commercially available, most of the biosensors and microarray based applications are still on the level of prototypes. Other promising methods with future potential include, Flow cytometry, Bacteriophage technology, biosensor based technology ect. 28.7.1.1 Rapid Biochemical Identification Techniques The identification of microorganisms with conventional methods using different biochemical tests is labour intensive, with high usage of consumables and reagents. Miniaturized systems in the form of biochip based technology and diagnostic kits have been developed that are based on the use of dehydrated growth substrates, or on ready-to use media. These techniques usually termed modern biochemical identification techniques, are widely used and have a high importance for daily laboratory practice. Pure cultures are grown in a variety of liquid or solid media and detected by a colour change, gas formation or enzyme induced changes of the colour of the substrate. The results can be compared to a diagnostic chart, or analyzed with a database. A number of diagnostic kits are in the market for the identification of pathogens, spoilage organisms, starter cultures, etc. The systems offer visual analysis of the results or databases might be used to interpret and analyze the results in semi-automated and automated systems. In a comparison with conventional methods the use of kits was considered accurate, efficient, time-, space-, labour-saving and cheaper than conventional methods. They have a

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widespread use and conventional methods are often mainly relevant for reference testing (Ronald, 2001). 28.7.1.2 Antibody-Based Methods Immunochemical analysis is a well-established technique with application to many different fields. Antibody-based methods have proven to be simple, rapid and sensitive for detecting and quantifying different types of food contaminants. Antibodies can be used to detect pathogens and spoilage organisms such as viruses, bacteria and moulds, but also low molecular weight food contaminants, such as mycotoxins, pesticides and veterinary drugs. However, the development has been constrained by the availability of high performance antibodies against the selected target analyte. The specificity of an immunoassay is largely determined by the intrinsic specificity of the antibody used for detection (Kerr et al., 2001). There are three different types of antibodies available for assay development: polyclonal antibodies, monoclonal antibodies (Kerr et al., 2001) and recombinant antibodies. Recombinant antibodies are a very powerful new approach which offers many advantages. Also the emergence of molecular imprinting techniques opens possibilities for new applications. The selection of the appropriate antibodies allows the construction of tests with broad specificity narrow selectivity. Usually, an enrichment step is necessary. Tests can be single-use format like dip-sticks or done in miniplates, microplates or special formats. Only a few basic forms of antibody assay formats exist, but many different modifications are on the market. Both homogeneous and heterogeneous assays are widely used, but homogeneous assays are considered superior in their ease-of-use. The different assays format include latex-agglutination test, immunodiffusion test, ELISA, and immunochromatography. Some recently developed immunoassays make use of the growth of the pathogens in the actual assay (Roda et al., 2002). 28.7.1.3 Immunomagnetic Separation Immunomagnetic separation (IMS) techniques can be used to replace or supplement and speed up the enrichment step that is usually necessary before the detection of pathogens (Chapman et al., 1997). IMS differs from the assay format described above, as it is not a detection method in itself. It can be combined with different end-detection methods. Often, PCR assays which are directly applied to food have a significantly decreased sensitivity which is based on the unspecific inhibition of the DNA polymerase by different food components. Through removal of these inhibiting food components from the sample, PCR ready DNA is easily obtained and the sensitivity of the PCR assay can be improved. Moreover, PCR assays use very small sample sizes. IMS allows the concentration of the sample by a factor 5–10 and thereby increases the sensitivity of the assay. A technique called magnetic capturehybridization PCR (MCH-PCR) involves lysing the bacteria to release DNA and hybridization with pathogen- specific gene sequences using biotin labelled DNA

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probes. Following capture of the hybrids by streptavidin coated magnetic particles; the bound DNA is used for PCR amplification. IMS has proven to be a useful tool to enhance the speed and sensitivity of different detection methods. All of the steps have currently to be performed by hand, but an automation of the method can be expected in the next future. 28.7.1.4 Nucleic Acid-Based Assays Nucleic acid-based assays can be either performed directly, or after amplification of the target sequence. Polynucleotides with a sequence complementary to the singlestranded DNA or RNA are used as probes. DNA sequences that are homologous to all members of the family of interest, or unique sequences that are particular to a species can be chosen for the construction of the probe. Probes that target different genera, species or different strains can thereby be constructed. Genes associated with virulence are often used for detection of pathogens. The ribosomal RNA represents an attractive target for probe design since it is present in multiple copies in most organisms, and databases with sequences for several thousand microorgansims are available. Ribosomal RNA contains stretches of conserved sequences interspersed with variable sequence regions, providing the scope to design singlestranded DNA probes for broad-range or specific target detection. The binding of the probe to the complementary cellular DNA/RNA is called hybridization. In order to detect hybridization, the probe needs to be labelled. Among others, Biotin, Digoxigenin and different fluorochromes have been used for labelling. Since there are many different assay formats available which are produced by many different manufacturers, only some will be introduced below. 28.7.1.5 Amplification-Based Methods Molecular assays have become faster, less expensive and through the development of homogenous assays easier to use, making them more attractive for routine use in food laboratories. There are a number of methods available that allow the amplification of the target sequence or the detection signal, including branched DNA technology (bDNA), Nucleic Acid Sequence-based Amplification (NASBA), the Ligase Chain Reaction (LCR), Transcription- mediated amplification, strand displacement amplification (SDA) and rolling circle amplification (RCA). PCR is now an accepted method to detect pathogens by amplification of the target DNA and detecting the target PCR products (Fenollar and Raoult, 2004). Detection of the amplification product has previously been done by gel electrophoresis, which is not a very convenient technique for routine use in a food laboratory. Recent advances have led to the development of homogenous assays for real-time detection which allow greater sensitivity and easy detection of PCR products that make PCR more suitable for routine-use in food laboratories (Poms et al., 2004). A major disadvantage of the PCR technology is that it will detect both viable and non-viable bacteria. The decreased sensitivity of the PCR reaction directly applied to a food sample is well known and can also lead to false-negative results. The inclusion of

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a short pre-enrichment step solves these issues, but also makes the analysis much slower. Several methods of sample preparation have been proposed to overcome this disadvantage including IMS, filtration, and centrifugation, the use of organic solvents and detergents and sample dilution. The use of intact RNA as target has been proposed to discriminate between viable and dead cells. The preparation and isolation becomes more difficult as RNA is much less stable. However, new specific isolation procedures for mRNA are currently being developed. The BAX system (Qualicon, Inc., Wilmington, DW. USA.) is the first commercial PCR system for the detection of food-pathogens (Bennett et al., 1998). The FSIS (Food Safety and Inspection Sevice) USA has recently adopted this system for screening of ready-to-eat food, meat poultry and egg products for Listeria monocytogenes. The BAX system works directly from an overnight enrichment of the target organisms without a separate DNA isolation step. All PCR reagents required for the test are incorporated into a single tablet. The principle of the TDF-PCR detection is based on an intercalating dye, SYBR green I, binds to the double-stranded PCR product. The assay is an in situ process, simultaneously amplifying the target DNA and directly detecting the increasing fluorescence signal during the annealing/elongation phase of the PCR reaction. One additional thermal cycle is run consisting of a denaturation step and a product annealing step. Fluorescence is monitored directly in the PCR reaction tubes during this final cycle. The rate of increase in fluorescence over time during the product annealing step yields a characteristic pattern for positive samples that can be differentiated from negative samples.

28.8 Future Technologies The most promising breakthroughs of the development of on-line or on-site, sensitive, low-cost, rapid methods for routine-use are expected to be made in the area of sensor technology. Many prototypes for food diagnostic application in the food and drink industry are currently being developed. They have high potential for automation and allow the construction of simple and portable equipment for fast analysis. These properties will open up many applications within quality and process control, control of fermentation processes, quality and safety control of raw materials, and for HACCP monitoring.

28.8.1 Microarrays Microarray analysis is an emerging technology that has the potential to become a leading trend in bacterial identification in food and feed improvement (Rasooly and Herold, 2008). Microarrays often are also referred to as biochip, DNA chip, DNA microarray or gene array, and allow conducting many analyses in parallel. The technology uses fluorescent-labeled probes amplified from bacterial samples that are then hybridized to thousands of DNA sequences immobilized on chemically

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modified glass slides. The whole gene or open reading frame(s) is represented by a polymerase chain reaction fragment of double-strand DNA, approximately 1000 base pair (bp) or 20–70 bp single-strand oligonucleotides. The technology can be used to identity bacteria and to study gene expression in complex microbial populations, such as those found in food and gastrointestinal tracts. Data generated by microarray analysis can be potentially used to improve the safety of our food supply as well as ensure the efficiency of animal feed conversion to human food, e.g., in meat and milk production (Al-Khadi et al., 2002).

28.8.2 Sensor Technology Sensor Technology covers a wide area of diverse techniques, including optochemical sensors and biosensors. Biosensors are a subgroup of chemical sensors where the analytical devices are composed of a biological recognition element such as enzymes, antibodies, receptors, proteins, oligonucleotides, or even a whole cell coupled to a chemical or physical transducer. A transducer measures the changes that occur when the sensor couples to its analyte. The sensitivity of the system is determined by the type of tranducers employed. Biosensors can be used for the detection of very different analytes such as pathogens, pesticides and toxins. Biosensors can be grouped according to their biological recognition element into immunosensors using antibodies and hybrid sensors using DNA or RNA probes. There have been many sensors developed for the detection of foodborne pathogens with the goal to overcome problems associated with traditional microbiological detection techniques such as time- and labor-intensive (Baeummer, 2003). In fact, biosensor advancements have greatly improved our ability to detect minute quantities of analytes as research into biosensors has mainly focused on detection platforms with very low detection limits (Rider et al., 2003). It has been estimated that 38% of reported pathogen biosensors in the past 20 years have been designed for the food industry. However, only a limited amount of methods are combined and currently exploited for their use in food diagnostics. As recognition elements, bioaffinity based receptors that use the selective interaction between ligand and receptor, antibody or nucleic acid are most widely used. As transducers, electrochemical and optical systems have gained practical importance. As nanobiotechnology progresses, sensors to detect pathogens or their constituents become smaller and more sensitive. Owing to the nature of these nanoscale sensors, the sample size from which the detection is being made is typically a microliter or smaller. Therefore, the challenge for scientists developing detection methods for pathogens in foods is in the sample preparation. Although the sample preparation requirements will vary from one food product to another, research into this step is required to bridge the emerging field of nanosensors with the food industry. Thus, while the organism with the largest number of diagnosed cases may fluctuate from year to year, the food industry will always be looking for detections system that will help identify all pathogens of concern in its food products (Rider et al., 2003).

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Optical biosensors have been developed for rapid detection of contaminants in foods, including pathogens, and several have evolved into commercial prototype systems. The analyte in the food interacts with the bioactive molecule, usually an antibody. Antibodies can be immobilized directly on the fibre, either on the blunt end or along the sides of a fibre tip. The binding of antibody and analyte is detected as a change in an optical signal measured through the fibre-optic assembly. The light from a laser travels to the fibre tip and penetrates into the area outside the tip. A fluorescently labelled complex binds to the antibodies on the tip. The fluorescent signal then radiates in all directions, and some of it travels back up the fibre tip to the detector. Detection of molecules in solution can be made either by direct binding to the biosensor coating molecules, or by competition binding with soluble capture molecules added together with the sample. Future applications might include protein quality and the detection of allergens genetically modified (GM) proteins, Bovine Spongiform Encephalopathy prions, pathogens and biocide residues.

28.8.3 Flow Cytometry Flow cytometry has mostly been used as a research tool for the study of microorgansims. However, the development of cheaper instrumentation should make it more useful for routine applications in the future. Flow cytometers can also be equipped with cell sorting devices allowing to sort out cells with a certain profile. By this method, Staphylococcus aureus could be separated form E. coli to a purity of 95%. The technique can provide rapid information on live and dead cells and information on their physiology and enables both qualitative and quantitative analysis of microbial cells in liquids. Due to the complexity and inherent cost of the equipment, the practical use of the method is still limited to research. However, with the appearance of cheaper systems on the market, it is considered to be a promising technology for the future.

28.8.4 Bacteriophage-Based Techniques Bacteriophages allow discrimination between different bacterial strains and this has been exploited in “phage typing” of bacterial samples. Recently, a chemiluminiscence based method for detection of food-borne pathogens has been developed. The bacteriophages are engineered to carry the luxAB gene, expressing the enzyme luciferase. The infected cell will produce light that can be measured with a photodetector. Viability is essential for the light production, so that the assay can discriminate between viable and non-viable cells. The assay is more rapid due to the faster replication cycles of the virus compared to bacteria, generating a 20–100 fold increase of the virus particle within 30 min. At present, there are no commercial kits available using this technology.

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28.8.5 Riboprinting and Pulse-Field Gel Electrophoresis Riboprinting and Pulse-field gel electrophoresis are used for the genetical characterization of microbial cells. These methods are able to produce characteristic DNA finger printing patterns that can be used for surveillance programmes for food borne diseases. By comparing patterns collected in central databases the sources of contaminations can be traced even for geographically separated incidents. The use of these methods is currently limited to research laboratories. Many contract research organizations (CROs) offer the application of these methods for detection of contamination sources as a service to the food industry.

28.8.6 Applications and Limitations of Rapid Methods The field of rapid methods is growing fast with numerous analytical options being available for different testing situation. These new rapid methods have the potential to offer considerable advantages regarding their sensitivity, rapidity and potential for automation, allowing increased sample through-put for analytical laboratories, and ease-of use (Magliulo et al., 2006; Lukinmaa et al., 2004). Most of the current rapid methods detect single target, but more and more methods with multiflexing properties are developed. Rapid methods are well suited for screening purposes for large numbers of food samples. Since most methods lack the required sensitivity, culture enrichment is still necessary before analysis. Benefits of enrichment include dilution of inhibitors, recovery of sub-lethally damaged cells and differentiation between viable and non- viable cells.

28.9 Business Outlook of Food Diagnostics Food diagnostics as a business area is relatively new and can still be characterized as an emerging one, partly because the industry and the market is not well defined. The business of food analysis is expected to grow through the increasing demand for food safety; however, it is unclear whether this will lead to actual market growth of food diagnostics. Especially in Europe, the growing political interest into food safety issues has been driven by a need to re-establish consumer confidence into the public food safety system, which was badly disrupted as a consequence of recent food contamination incidents, e.g. Bovine Spongiform Encephalopathy, enterohemorragic Escherichia coli, multi-drug resistant strains, Dioxin, and GMOs. At this stage it is not clear what kinds of business models will prevail as profitable and viable, because it is dependent on whether we are concerned with food analysis, food diagnostics, microbiological testing, or microbiological food testing. In terms of trends and needs it is, however, clear that food safety has become a public issue. This need is most visible in the growing public concern, i.e. consumers and political actors. This is not to say that the corporate community is unresponsive to this need, but there is an evident conflict between what can be done with respect to what is economically viable and what is publicly desirable. It is expected by the public that more testing,

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especially for food pathogens, will result in an increase of food safety of the product. However, the concept of food testing is limited in guaranteeing food safety and therefore the HACCP concept has been applied to guarantee process safety rather than product safety. There is a clear corporate and public communication challenge that needs to be addressed. The question of whether implementation of rapid methods will lead to a sufficient degree of improvement in food safety or will have more impact on cost savings has to be further analyzed (Glynn et al., 2008). It is not clear whether the consumers in general are willing to pay a premium price for food safety tests, on something which they already assume to be safe. Therefore, despite the growing public concerns over food safety, the economic incentives to develop new rapid methods are not exactly compelling. However, it is obvious that food safety has a very high impact on the image and branding of food processing companies and thus has a clear long-term corporate profit impact. In light of that it might be better to consider the issue of food safety and food diagnostics as an element of corporate communications. The business area of food diagnostics is currently a derivative of the much larger medical diagnostic industry and other diagnostic areas (veterinary, environmental, agricultural), because new technologies are developed there, which has implications for both technology and market management in terms of economies of scale and scope. There has been an increased and renewed interest in the US after September 11, 2001 to develop new methods for pathogen testing resulting in an increase of R&D funding in that area. Food diagnostics in terms of market attractiveness is competing with other potentially more attractive markets such as pharmaceutical technologies and services measured in terms of market potential. Food diagnostics is a fairly small market in absolute terms, but also in comparison to the whole market for food analysis. There are approximately 50 established companies with some market leaders but no clear market dominator. The small market size has implications for market attractiveness, which may not provide incentives enough to develop new technologies or to adopt new technologies developed in, e.g. in vitro diagnostics to the special requirements for the food industry.

28.10 Conclusion Diagnostic technologies can be applied in a variety of different areas either directly or after modification thus seemingly widening the commercial potential. Although there are apparent synergies between the different diagnostic sectors at the R&D level, the over all benefits might be meager as the markets are dramatically different, requiring for example modified product development processes, an entirely different sales force, technology transfer, and are subject to different regulatory requirements. The food industry itself is not expected to increase their R&D expenditures, which in most cases is currently keeping pace with inflation. R&D activities listed by importance are new product development, product safety research, clinical trial or support, quality control, quality assurance, ingredient technology, process development, market research and nutritional research analysis. Areas of food science in

References

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order of importance are healthy food, functional food, food safety from a process perspective, development of natural food, food safety from an ingredient point of view, organic food, reduced fat food, and methods development for quality control. Food diagnostics is set on an ongoing evolution. All the detection technology will be a part of the food safety surveillance programme using the latest molecular diagnostic tools.

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Malorny, B., Paccassoni, E., Fach, P., Bunge, C., Martin, A., Helmuth, R., 2004, Diagnostic realtime pcr for detection of Salmonella in food. Appl Environ Microbiol 70:7046–7052. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., 1999, Food-related illness and death in the United States. Emerg Infect Dis 5:607–625. Naravaneni, R., Jamil, K., 2005, Rapid detection of food-borne pathogens by using molecular techniques. Med Microbiol 54:51–54. Niggemann, B., Rolinck-Werninghaus, C., Mehl, A., Binder, C., Ziegert, M., Beyer, K., 2005, Controlled oral food challenges in children – when indicated, when superfluous? Allergy 60:865–870. Nowak-Wegrzyn, A., Sampson, H.A., 2006, Adverse reactions to foods. Med Clin North Am 90:97–127. Nugen, S.R., Baeumner, A.J., 2008, Trends and opportunities in food pathogen detection. Anal Bioanal Chem 391:451–454. Poms, R.E., Anklam, E., Kuhn, M., 2004, Polymerase chain reaction techniques for food allergen detection. J AOAC Inter 87:1391–1397. Rasooly, A., Herold, K.E., 2008, Food microbial pathogen detection and analysis using DNA microarray technologies. Food borne Pathog Dis 5:531–550. Rider, T.H., Petrovick, M.S., Nargi, F.E., Harper, J.D., Schwoebel, E.D., Mathews, R.H., Blanchard, D.J., Bortolin, L.T., Young, A.M., Chen, J., Hollis, M.A., 2003, A B cell-based sensor for rapid identification of pathogens. Science 301:213–215. Roda, A., Mirasoli, M., Venturoli, S., Cricca, M., Bonvicini, F., Baraldini, M., Pasini, P., Zerbini, M., Musiani, M., 2002, Microtiter format for simultaneous multianalyte detection and development of a PCR-chemiluminescent enzyme immunoassay for typing human papillomavirus DNAs. Clin Chem 48:1654–1660. Ronald, C., 2001, McGlennen miniaturization technologies for molecular diagnostics. Clin Chem 47:393–402.

Chapter 29

Rapid Diagnostic Methods for Biowarfare

Abstract There is a great concern within the scientific and security communities on the ongoing revolution in Genomics research. Genomics revolution may be used to enhance detection, protection and treatment so that biological warfare agents are never used. Biological weapons have the greatest potential for lethality of any weapon. Biological weapons are accessible to all countries; there are few barriers to developing such weapons with a modest level of effort. The current level of sophistication for many biological agents is low, but there is enormous potential based on advances in modern molecular biology, fermentation, and drug delivery technology for making more sophisticated weapons. Keywords Bioterrorism · Bioweapons · Anthrax · Diagnostics · Mycotoxins · Vaccines

Biodefence

·

Genomics

·

29.1 Prologue The advent of biotechnology, namely in the fields of microbiology, molecular chemistry and genetic engineering have opened new ways for human race to propel itself to a better future. However, like all things, there is a darker, more sinister side to biotechnology. One “dark side” is the manufacture and proliferation of biological and chemical weapons. Biological warfare agents have gained attention in recent years. Only a few years ago bioterrorism was considered a remote concern but today it has reached the forefront of the public imagination following recent terrorist attacks around the world. Advances in biotechnology and genetic engineering may facilitate the development of potentially new and more deadly biological warfare agents. The ability to modify microbial agents at a molecular level has existed since the 1960s, when revolutionary new genetic engineering techniques were introduced, but the enterprise tended to be slow and unpredictable. With today’s more powerful techniques, infectious organisms can be modified to bring about disease in different ways. Many bioengineering companies (both U.S. and foreign) now sell all-in-one kits to enable even high school-level students to perform recombinant M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4_29,

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DNA experiments. The availability of free on-line gene sequence databases and analytic software over the Internet further simplifies and disseminates this capability. It is now possible to transform relatively benign organisms to cause harmful effects. Genetic engineering gives biological warfare developers powerful tools with which to pursue agents that defeat the protective and treatment protocols of the prospective adversary. Genetically engineered micro-organisms also raise the technological hurdle that must be overcome to provide for effective detection, identification, and early warning of biological warfare attacks.

29.2 Concept During the twentieth century, major military powers have established secret biological warfare programs driven by visions of an extraordinary weapon that could penetrate behind enemy lines and kill civilians (Guillemin, 2005). Some weapons not only inflict casualties but manage to strike considerable fear in the enemy (Arora, 2005). “Biological Warfare” (BW) is use of living pathogens to produce casualties in man or animals or damage to plants. A BW weapon may be intended to kill, incapacitate or seriously impede on an individual as well as entire cities or places. Civilians may encounter these agents during a terrorist or military attack, a riot control operation, or due to accidental exposure during industrial or agricultural use. Military personnel may be exposed during their active use in the battlefield or exposure to areas where the disease may be chronically endemic. A number of biological agents (Török et al., 1997) could be used in a terrorist attack, including anthrax, botulinum, salmonellosis, plague, smallpox, staphylococcal and streptococcal toxins, and the list of emerging pathogens is evolving rapidly. In spite of major successes against infectious diseases in the twentieth century, new infectious diseases have emerged and old ones re-emerged in recent decades in different parts of the world (John, 1996).The serious diseases that their causal agents produce could cause considerable morbidity and mortality if used in a terrorist attack (Henderson et al., 1999). Of the weapons of mass destruction, the biological ones are the most feared and bioterrorism has become one of the most vicious threats to civilized society in recent times. Biological weapons have been sporadically used for centuries. Before the twentieth century, the use of biological agents took three major forms: • Deliberate poisoning of food and water with infectious material (Török et al., 1997) • Use of microorganisms, toxins or animals, living or dead, in a weapon system (Polyak et al., 2002) • Use of biologically inoculated fabrics. Despite international regulations, there has been a global re-emergence of the threat of biological warfare. As many as 17 countries are suspected of either including or developing biological agents in their weapons programmes. In the past decade, a number of terrorist organizations with access to bioweapons technology have emerged. Current surveillance systems may be inadequate to detect biological

29.3

Characteristics of Biological Weapons

485

attacks. The onset of illness is often delayed, thus the timing and location of such an event may be extremely difficult to identify. We are unfamiliar with most of the agents of biological warfare and are ill-equipped to handle the consequences of such an attack. In addition, there is no apparent coherent policy to handle a biological terrorist incident. Given the enormity of what is possible in the event of a biological attack, one must be prepared to detect, diagnose, epidemiologically characterize and respond appropriately to biological weapons. Of the potential biological weapons, smallpox and anthrax pose the greatest threats (Rotz et al., 2002). The question of what disease-causing organisms might supplant classic biological warfare agents is critical to understanding future biological warfare threats. Biological warfare agents may emerge in two likely categories: man-made manipulations of classic biological warfare agents and newly discovered or emerging infectious agents that result from natural occurrences. The future likelihood of infectious agents being created for biological warfare purposes will be influenced by several technological trends, of which four of the most significant are: • Genetically engineered vectors in the form of modified infectious organisms will be increasingly employed as tools in medicine and the techniques will become more widely available. • Strides will be made in the understanding of infectious disease mechanisms and in microbial genetics that are responsible for disease processes. • An increased understanding of the human immune system function and disease mechanisms will shed light on the circumstances that cause individual susceptibility to infectious disease. • Vaccines and antidotes will be improved over the long term, perhaps to the point where classic biological warfare agents will offer less utility as a means of causing casualties. The goal of biodefense is to integrate the sustained efforts of the national and homeland security, medical, public health, intelligence, diplomatic, and law enforcement communities. Health care providers and public health officers are among the first lines of defense. In some countries private, local, and State (province) capabilities are being augmented by and coordinated with federal assets, to provide layered defenses against biological weapons attacks. The traditional approach toward protecting agriculture, food, and water: focusing on the natural or unintentional introduction of a disease being strengthened by focused efforts to address current and anticipated future biological weapons threats that may be deliberate, multiple, and repetitive.

29.3 Characteristics of Biological Weapons The revolution in biotechnology facilitates an evolution in the biological warfare threat. The revolution in biotechnology began in 1977 with the successful cloning of a protein using a synthetic, recombinant gene. Scientific and technologi-

486

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cal advances have facilitated the development of genetically engineered agents. The extreme lethality of biological warfare agents has long been known. The most lethal biological toxins are hundreds to thousands of times more lethal per unit than the most lethal chemical warfare agents. However, lethality is only one of many characteristics necessary to consider in the development, production, and employment of a biological warfare agent. Numerous characteristics need to be controlled for a highly effective biological warfare agent. Historically, the accentuation of one characteristic often resulted in the attenuation of one or more other characteristics, possibly even rendering the modified agent ineffective as a weapon. Advances in biotechnology, genetic engineering, and related scientific fields provide increasing potential to control more of these factors, possibly leading to the ability to use biological warfare agents as tactical battlefield weapons. • The potential types of novel biological agents (microorganisms) that could be produced through genetic engineering methodologies: • Benign microorganisms are genetically altered to produce a toxin, venom, or bioregulator. • Microorganisms resistant to antibiotics, standard vaccines, and therapeutics. • Microorganisms with enhanced aerosol and environmental stability. • Immunologically-altered microorganisms able to defeat standard identification, detection, and diagnostic methods.

It is noteworthy that each of these techniques seeks to capitalize on the extreme lethality, virulence, or infectivity of biological warfare agents and exploit this potential by developing methods to deliver more efficiently and to control these agents on the battlefield. Ideal characteristics of biological weapons targeting humans are of high infectivity, high potency, non-availability of vaccines, and delivery as an aerosol (Atlas 1998). Diseases most likely to be considered for use as biological weapons are contenders because of their lethality (if delivered efficiently), and robustness. The biological agents used in biological weapons can often be manufactured quickly and easily. The primary difficulty is not the production of the biological agent but delivery in an effective form to a vulnerable target. Diseases considered for weaponization, or known to be weaponized are discussed below. Ongoing scientific research into the functioning of disease organisms also should provide insights for the development of advanced medical defenses against new and emerging biological warfare threats. Current examples of infectious organisms that are attracting particular attention are hantaviruses; other hemorrhagic fever-causing agents, such as Ebola; and the bacteria invasive Group A streptococcus (commonly known as flesh-eating bacteria). The streptococcus example is illustrative. Once it is well established, the infection is very difficult to control with antibiotics. Although the natural form of this organism may not have significant potential as an aerosol threat agent, those seeking new infectious agents for military use could investigate its mechanisms of action.

29.3

Characteristics of Biological Weapons

487

29.3.1 Anthrax Anthrax is primarily a zoonotic disease caused by Bacillus anthracis that produces a toxin consisting of a protective antigen, edema factor and a lethal factor (Witkowski and Parish, 2002). Anthrax is considered an effective agent for several reasons. First, it forms hardy spores, perfect for dispersal aerosols. Second, pneumonic (lung) infections of anthrax usually do not cause secondary infections in other people. Thus, the effect of the agent is usually confined to the target. A pneumonic anthrax infection starts with ordinary “cold” symptoms and quickly becomes lethal, with a fatality rate that is 90% or higher. Finally, friendly personnel can be protected with suitable antibiotics. Diagnosis is based on gram stain, cultures or direct immunofluorescence or ELISA or PCR. Anthrax toxin may be detected in the blood by immunoassays (Polyak et al., 2002). A simple mAb based dot-ELISA is available for detection of toxin-producing strains of Bacillus anthracis (Shastry et al., 2003).

29.3.2 Plague Infection to Yersinia pestis has resulted in high mortality and thus become a potent BW agent. On inoculation 4–10% individuals may develop a pustule at the inoculation site. Terminal pneumonic and septicaemia plague patients would develop livid cyanosis and large ecchymoses on the back. Septicemia could cause petechiae, purpura, ecchymoses, and acral cyanosis and necrosis leading to the infamous description, “the Black Death.” (Inglesby et al., 2000). Investigations include bubo aspiration for direct microscopy, and culture on blood agar of bubo aspirate, cerebrospinal fluid, blood and sputum. PCR- and ELISA-based systems are also available for detection or strain typing of Y. pestis alone or in conjunction with virulence markers such as F1 (fraction 1) and Pla (plasminogen activator) (Khushiramani et al., 2009).

29.3.3 Tularemia Tularemia is a zoonosis caused by Francisella tularensis. The commoner forms include the ulceroglandular form, which involves the skin and lymph nodes, and the typhoidal form. A cutaneous ulcer occurs in approximately 60% of patients and is the most common sign of tularemia. Ulcers are generally single lesions of 0.4–3.0 cm in diameter, with heaped-up edges and accompanied with systemic upset. The organism is difficult to culture. Hence the diagnosis is usually established by serology. A live, attenuated vaccine is effective against aerosol infection (Friedlander and Evans, 1997).

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29.3.4 Melioidosis Burkholderia (formerly Pseudomonas) pseudomallei is a gram-negative bacillus. Cutaneous manifestations include severe urticaria flushing and cyanosis. Inhalational melioidosis could lead to pustules or cutaneous abscesses associated with lymphangitis, cellulitis, or regional lymphadenitis, but only after metastatic abscesses to the skin are formed, and this would be likely to take months (Wuthiekanun et al., 2004).

29.3.5 Viral Hemorrhagic Fevers Hemorrhagic fever viruses, with the exception of dengue, are potentially transmitted via aerosol. Cutaneous manifestations are non-specific and include flushing, petechiae, purpura, ecchymoses, and edema. ELISA to detect specific IgM antibody responses must be done. One should not wait for viral cultures, which consume time, to initiate management. Management includes supportive care for prevention of hypotension and shock. Medical personnel need to be protected. Isolation of the patient during active disease and strict barrier nursing must be adopted. Specific treatment may include ribavirin. Only yellow fever has a licensed vaccine (Barrett, 2004).

29.3.6 Other Viral Fevers The viral species important from the warfare context include variola (smallpox), monkeypox, and vaccinia. Smallpox has an incubation period of 7–17 days and a prodrome of 2–4 days. An enanthem on the buccal and pharyngeal mucosa starts on about the second day. An orderly progress of macules to papules, vesicles, pustules (sometimes umbilicated), and crusts distributed in a centrifugal pattern (in distinction to varicella) over a 1–2 week period is typical. Crusts are infective and detach in about 3 weeks leaving depressed, hypopigmented scars. Monkeypox greatly resembles variola, the only distinguishing feature being cervical and inguinal lymphadenopathy.

29.3.7 Trichothecene Mycotoxins Trichothecene mycotoxins (“Yellow rain”) are the only potential BW toxins with cutaneous activity and manifestations. Mycotoxins are a diverse group of small molecular weight compounds produced by the fungi of five fungal genera: Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps. “Yellow rain” was used in Southeast Asia between 1974 and 1981. Most attacks used yellow pigment, but some used red, green, white, or brown smoke or vapor. Vesication often

29.4

Biodefense

489

occurred with “yellow rain” attacks. At low doses (nanograms), severe skin irritation with erythema, edema, and necrosis is observed (Seely et al., 1985). A mask and full-body clothing are protective. The clothing gear and contaminated areas of skin should be washed with soap and water followed by a water rinse. Washing within 4–6 h of exposure removes 80–98% of the toxin and prevents death and dermal lesions in experimental animals. High doses of systemic steroids decrease toxin injury.

29.3.8 Aflatoxin This is a class of toxin, produced by Aspergillus flavus that induce liver cancer. Since there is difficulty in differentiating cancer origins between accidental and intentional exposure, even recognizing that a target population had been “attacked” would be laborious; this would be a case of a “stealth BW attack”. 29.3.8.1 Ricin Ricin is a protein toxin extracted from the castor bean plant and exhibits toxic effects on cells of different visceral organs (Kumar et al., 2007). Ricin is already being investigated for its “magic bullet” properties as an agent that might selectively destroy cancer cells (Rao et al., 2005). That same principle could be used to specifically target an enemy; in theory one could be specific enough to use this procedure to target a single individual for assassination. 29.3.8.2 Fusarium oxysporum The potential use of genetic engineering in the production of BW is illustrated by the on-going studies on the possible use of the mould Fusarium oxysporum as a candidate for drug plant eradication. This fungus, which has devastated commercial crops (e.g. bananas and muskmelon), is being investigated for its potential to destroy coca and cannabis plants. Preliminary studies indicate that host specificity is narrow and species “jumping” is rare; i.e., targets can be carefully selected without posing danger to other commercial crops. Obviously, the same technology could be applied by terrorists to assail the commercial crops of perceived enemy states.

29.4 Biodefense It is important to note that all of the classical and modern biological weapons organisms are animal diseases, the only exception being smallpox (Henderson et al., 1999). Thus, in any use of biological weapons, it is highly likely that animals will become ill either simultaneously with, or perhaps earlier than humans. Indeed, in the largest biological weapons accident known the anthrax outbreak, sheep became ill with anthrax as far as 200 km from the release point of the organism from a

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military facility in the southeastern portion of the city.Thus, a robust surveillance system involving human clinicians and veterinarians may identify a bioweapons attack early in the course of an epidemic, permitting the prophylaxis of disease in the vast majority of people (and/or animals) exposed but not yet ill.

29.4.1 Viral Agents – Poxviridae Poxviruses, the largest of all viruses, differ from other DNA viruses by replicating in the cytoplasm where they produce eosinophilic inclusion bodies. They are relatively resistant to drying and many disinfectants. The Orthopox genus includes at least nine species.Three viruses interest us in a BW context: variola, monkeypox, and vaccinia. Variola is an orthopox virus very similar to vaccinia but with different host predilections. There was no animal reservoir (although monkeys are susceptible to infection); this factor enabled global eradication of this disease. Variola retains its transmissibility for one year in dust and cloth. Person-to-person transmission requires close contact. Patients were most infective 4–6 days after the illness started, and respiratory spread was probably the most common route of transmission. Only 30% of susceptible contacts became infected. Monkeypox was first identified in 1958 as a pathogen of cynomolgus monkeys; in 1971 it was linked to human disease. The virus exists in an enzootic state in arboreal squirrels of tropical rain forests of western and central Africa. Person-to-person transmission by respiratory droplet occurs.

29.4.2 Botulinal Toxins Botulinal Toxins (botox) are produced by a microscopic organism Clostridium botulinum. Clostridial neurotoxins are among the most toxic substances known and cause severe illnesses in both humans and animals (Dixi et al., 2006). A deadly poison even in small doses. It can be absorbed through the skin, lungs, eyes and mucous membranes. The poison occurs in the soil and naturally in low acid foods. It can be distributed through aerosol methods, water and food. The symptoms are delayed (2– 14 days), and the irreversible damage is done (walking dead) before victims realize what has occurred (Arora et al., 2002). A vaccine for the disease is available but to obtain complete protection from the toxins, the amount needed would cause death so it is not usually recommended, except to certain military personnel. If administered within 48 h from exposure, an antitoxin can only stop the progress of the disease. Oxygen should also be provided for assisted breathing. Formaldehyde kills botulinum spores. Boiling in water for at least 10 min will kill the poison, not the spores. To kill the spores, it takes more than 6 h of boiling in water.

29.5

Combating Detection of Biowarfare Agents

491

29.4.3 Mycotoxins Trichothecene Mycotoxins are produced by fungi of the genera Fusarium, Myrotecium, Trichoderma, Stachybotrys and others. They inhibit protein synthesis, impair DNA synthesis, alter cell membrane structure and function, and inhibit mitochondrial respiration. The toxins, protein in nature, have a low molecular weight. They also contain food refusal and emetic factors. Trichothecene mycotoxins are highly persistent and stable for long periods of time. 0.5 mg of the poison is enough to kill half the exposed humans. The skin of the victims can be irritated if the skin is exposed to the toxins. They can also cause radiomimetic injury of intestines, bone marrow, lymph nodes, spleen and thymus, leading to leukopenia and bone marrow atrophy. Effects are also found on central nervous, circulatory and reproductive systems. After about 8 weeks from exposure to Normocyclic Trichothecenes, one will suffer from Alimentary Toxic Aleukia; burning sensation in the alimentary tract, vomiting, tachycardia, leukopenia, petechial hemorrhages with necrosis in skin and internal hemorrhages. After about 8 weeks from exposure to Macrocyclic Trichothecenes, one will suffer from Stachybotryotoxicosis, conjunctivitis, rhinitis, leukopenia, dematis and pulmonary fibrosis (Table 29.1).

29.5 Combating Detection of Biowarfare Agents A growing demand for methods to detect the presence of biological warfare (BW) agents and other pathogens in samples from the environment, the battlefield, and food is currently driving a need for new detection technologies. Especially for biodefense and risk evaluation, an early and definite technology is needed. A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents.

29.5.1 Prophylatic & Therapeutic Approaches Traditionally, vaccines consisted of a preparation of the infectious agent itself – either living, weakened or killed. Introducing the vaccine into the body activates the immune system, resulting in the production of antibodies against that particular agent. If a vaccinated person is later exposed to the infectious agent, he or she will already have built up immunity against it. More recently, researchers have started using fragments of the pathogen’s DNA genome as a vaccine, rather than the entire organism. This approach helps eliminate the risk of infection that comes with using traditional vaccines. Certain natural products and anti-oxidants are found highly promising against chemical as well as biological warfare agents (Vijayaraghavan et al., 2006).

Fever, malaise, cough, respiratory distress High fever, chills, headache, productive cough – watery then bloody Dry throat, blurred vision, slurred speech, difficulty swallowing, progressive descending symmetrical paralysis Non-specific flu-like prodrome (malaise, fever, headache) then synchronously evolving maculopapular rash progressing to vesicles then pustules Irregular fever, chills, headache, malaise, cough and chest pain in 20%, osteoarticular disease

Inhalation anthrax

Brucellosis

No

High

No

High

No

10–100 organisms

Assumed low (10–100 organisms)

0.001 ng/kg is LD50 for type A

40 years High unless For up to 1 treated within year in soil; 12–24 270 days in hours live tissue Death in 24– High without For weeks in 72 hours; lasts respiratory non-moving month if not water and support lethal food

3–5 days (usually fatal if untreated) 1–6 days (usually fatal)

Duration of illness

Doxycycline + Rifampin

Cidofovir

Antitoxin supportive care

Streptomycin Gentamycin or Chloramphenicol

Ciprofloxacin Doxycycline

Treatment

29

Smallpox

Botulism

Pneumonic plague

Symptoms

Disease

Person-toperson transmission

Table 29.1 Selected biowarfare agent characteristics

492 Rapid Diagnostic Methods for Biowarfare

Fever, headache, malaise, weight loss, nonproductive cough Fever, chills, headache, diaphoresis, malaise, fatigue, anorexia, and weight loss

Tularemia

Viral Fever, rigors, severe Encephalitides headache, photophobia, malaise, nausea, vomiting, diarrhea may follow Viral Fever, malaise, Hemorrhagic myalgia, prostration; Fevers vascular permeability may present as conjunctival injection and petechial hemorrhage and progress to mucous membrane hemorrhage and shock

Q Fever

Symptoms

Disease

10–100 organisms

1–10 organisms

Moderate

1–10 organisms

10–50 organisms

Infective dose (Aerosol)

Low

Rare

No


4–21 days

1–5 days

3–6 days (range 1–21 days) 7 days (range 2–14 days)

Incubation period

Days to weeks

Days to weeks

Weeks

2 weeks

Duration of illness

Table 29.1 (continued)

5–90% case fatality rate depending on virus

Variable

Very low

Moderate if untreated

Lethality

Treatment

Relatively Ribavirin unstable in the Supportive care environment

For months in Streptomycin moist soil or Gentamycin other media Able to Tetracycline withstand heat Doxycycline and drying; persists in environment for weeks to months Relatively Supportive unstable in the care environment

Persistence of organism

29.5 Combating Detection of Biowarfare Agents 493

Symptoms

Staph Sudden onset fever, Enterotoxin B chills, headache, myalgias, non-productive cough Ricin Depends on route of exposure. Aerosol route: fever, chest tightness, cough, hypothermia. Oral route: gastrointestinal hemorrhage T-2 Mycotoxins Skin pain, redness, necrosis, sloughing of epidermis, wheezing, chest pain, hemoptysis

Disease 30 ng/person incapacitation

3–5 g/kg is LD50

Moderate

No

No

Infective dose (Aerosol)

No


Variable. Death may occur in minutes, hours or days

Days. Death within 10–12 days for ingestion

Days

Duration of illness

Moderate

High

70%) of this market. Other market segments include traditional genetics, personalized medicine, and cancer with 13, 9, and 8% of the U.S. clinical labs services market, respectively. Because infectious diseases are a mature segment, these other categories are growing more rapidly on a relative basis. Molecular diagnostics is also starting to emerge as an important tool for inherited disorders, cardiovascular disorders, and other disease areas. The driving force behind the rapid expansion of the diagnostic market can mostly be attributed to explosive developments in immunologic and DNA technologies propelling both new and established companies forward. Biochip and nanobiotechnology are expected to make a significant contribution to the growth of molecular diagnostics. Recent developments in the field of Pharmacogenomics are expected to make rapid developments in making disease diagnosis and therapy tailored to an individual’s genetic makeup (often referred to as personalized medicine). Almost all of the Molecular Diagnostics tests today are based on PCR, but other technologies (most notably microarrays) are taking market share away.

30.5

Trends in Cancer Diagnostic Testing World Markets

507

30.4 Trends in Infectious Diseases Testing Market Diseases caused by bacteria, viruses, fungi and other parasites are major causes of death, disability, and social and economic disruption for millions of people. Between 14 and 17 million people die each year due to infectious diseases – nearly all live in developing countries. In developing countries, lower respiratory infections, HIV/AIDS, diarrheal diseases, malaria and tuberculosis are among the top 10 causes of death. Early and sensitive detection of viral and bacterial infections is currently achieved by PCR based amplification technologies. Viral load testing has become standard for management of HIV, hepatitis C virus (HCV), and human cytomegalovirus infections, while nucleic acid probe-based testing for HIV and HCV is now in general use in specialized clinics and hospitals. The first molecular diagnostic tests to reach the market were infectious disease tests, and are one of the most rapidly growing segments of the in vitro diagnostics industry. This segment is dominated by tests for HIV, sexually transmitted diseases and hepatitis and this remains the largest segment of the molecular diagnostics market. Among the main driving forces behind the growth of this dynamic market is the spread of AIDS, which remains the world’s major health threat and the key factor contributing to the rise in incidence of opportunistic infections. In 2004, the infectious disease diagnostics segment contributed up to 80.0% to the European molecular diagnostics markets, indicating high end-user acceptance of them. New analysis from Frost & Sullivan finds that the U.S. Infectious Disease Diagnostics Markets, as defined in the research service, earned revenues of $3.8 billion in 2005 and estimates this to reach $6.4 billion in 2012. The segment is expected to grow at an annual rate between 7 and 8% over the next few years and will present some excellent market opportunities for molecular diagnostic competitors.

30.5 Trends in Cancer Diagnostic Testing World Markets Cancer is the second most common cause of death after cardiovascular disease in developed countries and cancer diagnostics is a growing market due to the ageing population. Cancer testing is one of the most important growth opportunities for the next three to five years in the diagnostics segment. Currently, cancer diagnostics market consists mainly of methods based on immunodiagnostics for detecting tumor markers. A number of companies are developing predisposition tests based on genomic variations associated with earlier stages of cancer. Co-development of molecular diagnostics and targeted therapeutics has already been proven to be a successful strategy in the development of novel anti-cancer drugs. Pharmacogenomic tests that permit tailoring chemotherapeutic therapy to the genetic characteristics of an individual patient and tumor have began to appear on the market. The unraveling of human genome is enabling biomedical scientists to workout molecular signatures of different cancers which may permit earliest possible pre-symptomatic diagnosis. A near-term market opportunity for cancer companion diagnostic tests exists

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Segments of Molecular Diagnostics – Market Place

in drug selection for cancer therapy. The National Cancer Institute estimates that about ten million Americans have or have had some form of cancer. Traditionally cancer has been treated with surgery, chemotherapy, hormones and radiation therapy, alone or in combination. Emerging technologies include photodynamic therapy, gene therapy, and immunotherapy and angiogenesis inhibitors. Pharmaceutical and biotechnology companies are investing billions of dollars to search out and develop weapons for the arsenal in the war against cancer. Overall costs of the disease are $126 billion annually. Pharmaceutical companies are developing more than 300 new medicines for cancer, some of which are in development for more than one type of the disease, for a total of more than 500 ongoing R&D projects. Molecular oncology tests have been emerging as a second significant – and rapidly growing – market segment. One additional infectious agent, human papillomavirus, has joined the list of major tests. Cancer and pharmacogenomic diagnostics generated 10.0% of revenues in 2004.

30.6 Trends in Cardiac Marker Diagnostic Testing Markets This immunoassay segment growth will result from tests based on recently validated biomarkers. One such growth opportunity example is in the cardiovascular disease drug therapy area where, currently, the relevant tests are only worth a few percent of the IVD market. Most tests relating to heart disease, e.g., cholesterol and triglycerides are done on high-throughput clinical chemistry analyzers using small volumes of inexpensive reagents. Thus the large numbers of cardiovascular tests performed annually worldwide do not translate into large markets. However, sales of the newer cardiac markers, which are immunoassays, are growing strongly. European growth rates in 2003 exceeded 35%. These molecular diagnostics, used in risk stratification and therapy monitoring, illustrate the increasing role of specific biomarkers in selecting and guiding drug therapy.

30.7 Point of Care Diagnostic Testing World Markets Point of care diagnostics is one of the most dynamic parts of the diagnostics industry and an area where several major competitors are battling for market share. Point-ofcare testing (POC) is defined as diagnostic testing at or near the site of patient care. Over the years, the increasing introduction of portable and handheld instruments has resulted in the migration of POC testing from the hospital environment to a range of medical environments including the workplace, home, disaster care and most recently, convenience clinics. A recent market survey of US hospitals indicates significant growth in the number of hospitals performing point-of-care testing. Several established hospitals across the globe, now uses point-of-care testing for glucose, urinalysis, fecal occult blood, gastric occult blood, blood gases, provider microscopy, pregnancy, rapid cardiac markers, rapid strep and rapid influenza.

30.8

Next Generation Molecular Diagnostics

509

Growth in point-of-care testing will come about as more tests become available such as a new point-of-care technology for continuous non-invasive monitoring of blood gases and electrolytes during surgery. This system replaces repetitive testing by the central laboratory which can provide only intermittent results. In addition, the rapidly expanding market for home testing and patient self-monitoring will continue to drive growth. The POC testing market is currently estimated to be worth US$11.3 billion in 2007 and is growing at 11% a year. POC testing accounts for approximately 34% of the US$33.6 billion global IVD testing market. Diagnostic testing accounts for only between 1and 2% of government healthcare expenditures worldwide, yet influences between 60 and 70% of healthcare decisions. Unsurprisingly, the POC market has also seen moves by large multinationals which previously had no presence in the IVD market. Siemens, for example, made major acquisitions to establish its diagnostics division, with Diagnostics Products Corporation and Bayer Diagnostics coming under the Siemens umbrella in 2006 at a cost of US$5.7 billion. The company has also just completed the acquisition of leading diagnostics company, Dade Behring, in a transaction valued at approximately US$7 billion. It is believed that Over-the-Counter Diagnostic Products World Markets market segment has the potential for growth rates above 15% per year over the next five years in some of the less well-explored and well-developed areas.

30.8 Next Generation Molecular Diagnostics The current molecular diagnostics products consist mainly of tests designed around a single biomarker associated with a disease state. Next-generation molecular diagnostics will employ multiplexing platforms, such as DNA or protein microarrays, to perform parallel biomarker analyses. Increasing automation and miniaturization, combined with new technologies that allow ultrasensitive quantitative multiplexed detection, is expected to increase sensitivity and reduce cost for many diagnostic applications. There are indications that the next advance will involve measuring several biomarker types simultaneously (for example DNA and protein). Such innovations relate to constructs, such as DNA/antibody microarrays capable of assaying proteins and NATs simultaneously, or to immunoassays using nucleic acid labels, where the results are amplified and read via PCR. Such tests would allow immunoassays and nucleic acid tests to be performed on a single instrument. Also observed is a growing trend from low- to high-margin molecular diagnostics as developers have become more concentrated and have capitalized on patent-protected protein and gene-based biomarkers. This should make investment in molecular diagnostics more attractive to investors. Innovation in the molecular diagnostics area appears to be robust as demonstrated by an analysis of issued patents and published patent applications from January 2000 to September 2008.

510

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30.9 The Rise of Companion Diagnostics The term companion diagnostic means that the particular diagnostic test under evaluation is specifically linked to a known therapeutic drug. This linkage could be important in the therapeutic application and clinical outcome of a drug (personalized medicine), or an important component of the drug development process. Pharmacogenomics, though still developing, holds immense potential to develop into a lucrative market as the demand for personalized medicine increases. Many drug companies are evaluating business models based on molecular diagnostics for pharmacogenomic testing as the driving force behind the shift toward personalized medicine. A number of hurdles confront the personalized medicine model, including regulatory policy, patient-provider cost coverage, and patient record privacy.

30.10 Market Considerations and Forecasts The number of companies involved in molecular diagnostics has increased remarkably during the past few years. More than 500 companies have been identified to be involved in developing molecular diagnostics and 250 of these are profiled in the report along with tabulation of 573 collaborations. The molecular diagnostic segments are projected to outperform the overall diagnostics market, growing from $13.8 billion in 2005 to $22.7 billion in 2010 (AGR of 10.4%), in contrast to the overall forecasted increase from $36.5 billion in 2005 to $53.6 billion in 2010 (AGR of 8.0%) for the entire IVD market. Although the overall laboratory-based immunoassay segment is a mature market, the molecular diagnostics segment of the immunoassay market of $12.8 billion in 2005, is expected to increase at an AGR of 8.9% to $19.5 billion in 2010. The sales of the newer cardiac markers, which are immunoassays, are growing strongly. European growth rates in 2003 exceeded 35%. The first generation of marketed molecular diagnostics has been based on established immunoassays and nucleic acid testing (NAT) technologies. Although the NAT market continues to be dominated by Roche, Abbott Laboratories and Bayer, many biotechnology companies have recently acquired significant intellectual property rights in this area. The company leaders in immunoassay and NAT identified in the D&MD Report include Roche Diagnostics, Abbott Laboratories, and the Ortho Clinical Diagnostics and Lifescan units of Johnson & Johnson, Beckman Coulter, Bayer Diagnostics, Becton Dickinson, Dade Behring, and bioMerieux. Important niche suppliers include Agendia, Bio-Rad, Biosite Diagnostics, Cepheid, Chembio Diagnostic Systems, Chiron (Novartis), Ciphergen Biosystems, Correlogic Systems, Cytyc, DiagnoSwiss, Diagnostic Products, Digene, DiaSorin, Epigenomics, EXACT Sciences, Gen-Probe, Genaissance Pharmaceuticals, HandyLab, Innogenetics,

30.10

Market Considerations and Forecasts

511

Interleukin Genetics, Matritech, Myriad Genetics, Nanogen, Nuvelo, Nymox Pharmaceutical, OXIS International, Proteome Sciences, Sequenom, SomaLogic, Sysmex, and Third Wave Technologies. Roche (40% market share), Gen-Probe, and Chiron lead the market with their dominant positions in infectious disease and blood-screening areas. The top nine players (Roche, Chiron, Gen-Probe, Abbott, Digene, Bayer, Myriad Genetics, Becton Dickinson, and bioMerieux) own approximately 75% of these markets. Leon projects that the cancer segment is growing at about 20% a year, traditional genetics about 15% a year, and personalized medicine about 20% a year, compared to the 5–10% growth rate for infectious diseases. Growth also varies by individual tests within segments. For the Digene HPV test, the company reported a 38% increase in sales from fiscal 2005 to 2006, or $97.4 million to $134.4 million, then a 42% growth rate for the first nine months of fiscal 2007 compared with the same period in 2006. The company has since been acquired by Qiagen. For the Oncotype DX test, Genomic Health reported revenues of $4.8 million in 2005, $27 million in 2006, and $27.7 million for just the first six months of 2007. In a recent web survey, over 250 participants ranked the segments poised for growth over the next two years and the next five years. Many of the survey respondents work in research areas such as biomarker discovery and validation, diagnostics research, diagnostics product development, discovery or preclinical research, and clinical research. The responses hence reflect more of the R&D phase rather than already commercialized products. Near term, respondents predicted significant growth in oncology at 33% and infectious disease at 30.1% with less growth seen for genetic testing at 14.5%, and pharmacogenomics at 12.5%. Longer-term growth, as predicted by the survey respondents, remained the same categorically, but priorities changed. Increase in the oncology market was about the same at 34%, the pharmacogenomics prediction increased to 26.5% long term, infectious diseases growth over the long haul fell to 14.6%, and genetic testing was also forecasted to see less growth at just 12.3%. In vitro molecular diagnostics have established themselves as effective tools for all aspects of disease management, especially in areas of unmet clinical need. Such tests have been developed for screening and prognosis as well as for applications, such as determination of genetic predisposition to disease, detection of presymptomatic disease, and prediction of individual drug response. Molecular diagnostics are the basis of pharmacogenomics, enabling the evolution of personalized medicine. Current molecular diagnostics are primarily single-analyte tests involving the detection of a single gene or protein. However, many disease-related processes are multifactorial, involving the abnormal expression of multiple genes or proteins. Second-generation molecular diagnostics are anticipated to utilize novel detection technologies and multiplexing platforms to allow the measurement of a large number of analytes simultaneously. These innovations will increasingly utilize multiplexing platforms such as DNA microarrays that perform parallel biomarker analyses. Regulatory events of note during the last two years include FDA approval of the first DNA microarray instrumentation system for in vitro diagnostic use

512

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(Affymetrix’ GeneChip System 3000Dx) and the first highly multiplexed diagnostic microarrays: Roche’s AmpliChip CYP450 test and two cystic fibrosis tests – Tm Bioscience’s Tag-It and Osmetech’s eSensor. Although not yet approved by the FDA, tests based on transcriptomic profiling have also debuted successfully on the market. There will be a steady increase in demand for new diagnostic testing services in the next five-year period, along with pressures to improve the quality of healthcare delivered in the clinic and also to lower its costs. Molecular diagnostics is a rapidly growing and rapidly changing market. Our forecast annual growth rates for the leading application groups are: tumour markers 8%, speciality immunoassay markers 15%, NAT for infectious disease 21% (except genotyping applications, where we forecast 44%), cancer screening (NAT) 34%, genetic testing 10%, and predispositional/theranostic testing (NAT) 26%. Geographically, the markets for these tests overall will grow slowly in Japan, somewhat faster in the US, and relatively rapidly in Europe. With in a span of 2–3 yrs both the US and Europe will each have around 35% of the worldwide diagnostics market. US demand for in vitro diagnostic (IVD) products will grow 5.1% annually through 2011. Clinical chemistry and immunoassay methods will remain the top two IVD sciences, while nucleic acid testing products will continue to generate the fastest gains. Hospital labs will stay the largest market while home health care will grow the fastest. With the exception of blood screening, adoption of molecular diagnostics by the rest of world and Japan is limited to the infectious disease segment. Faster growth rates and therefore superior commercial opportunities are expected in few developing regions. The major suppliers of established immunoassay systems have proved able to leverage their experience in dealing with clinical laboratories in order also to become leading suppliers of NAT products. Such companies include Roche, Abbott, and Bayer, which have at least 30% of the market between them. The Molecular diagnostics segment is increasing at a rapid rate for two reasons viz., introduction of early-stage, new cutting edge diagnostic technologies by major market players and secondly due to rapid development of automated systems and throughput technologies. Studies indicate that molecular diagnostic companies are expecting continued strong growth. Many of today’s molecular diagnostic tests are complex and low-volume compared to other traditional in vitro diagnostic tests and is only performed by 25% of the clinical laboratories that perform in vitro diagnostic tests. However, as test volumes increase and automation improves, molecular diagnostic tests will eventually be routinely performed by all segments of the clinical laboratory. As advances in molecular diagnostics continue, molecular diagnostic enterprises will become more attractive to venture capitalists, a group that previously stayed away from such investments. The wave of new information provided by molecular diagnostics has ushered in an era, evidenced by the growing importance of FDA guidelines on pharmacogenomics and the coupling of in vivo imaging techniques with molecular tests and molecular markers. Companies choosing to play in this space must understand its complexities and pitfalls.

References

513

References The information presented in this report is derived from publicly available information sources such as company, government, and medical organization reports and internet resources such as Molecular and Nucleic Acid Testing Market Reports from web based Kalorama information services. Avi Chaudhuri, 2008. Molecular Diagnostics: Future Potential for the Indian Marketplace, PAC Med Biotech Pvt. Ltd. Blood: The Worldwide Market for Blood Products, Blood Testing, Blood Equipment, and Synthetic Blood Products. Nov 1, 2008 – KLI1816262 Diagnostic Test Service Commercialization in Multiplex and Esoteric Testing: A Roadmap to Diagnostics in the 21st Century. Aug 1, 2008 – KLI1806317 Drug Discovery & Development. Magazine Vol. 9, No. 9, September 2007, p. 68. Glucose Testing Markets. Nov 1, 2008 – KLI1911068 Genetic engineering & Biotechnology News 2007, Vol. 27, No. 3 HIV: Markets for Diagnostics and therapeutics. Oct 1, 2008 – KLI1646600 Lab Automation Markets Worldwide (Next-Generation System, Key Companies, Forecasts and trends) Aug 1, 2008 – KLI1670001 Marena Subitra Rajeevan., 2006. Immunoassays versus Molecular Diagnostics: Who has the upper hand? Research Analyst, Healthcare. March issue. Retail Clinics and the In-Store Healthcare Market 2009: Bright Spot in the Current Economy? Mar 1, 2009 – KLI2041111 The World Market for Molecular Diagnostics. Jan 1, 2009 – KLI1914575 The Worldwide Market for In Vitro Diagnostic (IVD) Tests, 6th edition. Dec 1, 2008 – KLI1430266 The Clinical Laboratory Services Market (Growth Opportunities, Competitive Analysis and Competitor Profiles) Nov 1, 2008 – KLI1646590 Worldwide In Vitro Diagnostics (IVD) Market Research Bundle (Worldwide Market for In Vitro Diagnostic Tests, SNP Genotyping Markets, Companion Diagnostics, Testing Services Markets) Oct 1, 2008 – KLI1927625

Index

A Alzheimers, 201 Amplification, 7, 15, 38–39, 59–60, 130–143, 174–175, 217–218, 233–234, 238–241, 309–320, 340, 366, 387, 457, 460–461, 475–476 Amplification based test, 313 Analytes, 8, 20, 196, 211–212, 298, 385, 477, 513 Antemortem, 430 Anthrax, 6, 146, 175, 277, 390, 484–485, 487, 489, 492, 496–497, 506 Anthropometry, 431–432 Antibiogram, 231 Antibody, 19–20, 43–44, 51, 56, 65, 77, 137, 172–173, 175–177, 182, 184, 187–188, 190, 212–213, 216, 249, 276, 295, 301, 317–318, 320–321, 362, 387–388, 414–415, 473–474, 495, 511 Antibody-based microarray techniques, 175 Antibody based test, 19, 295, 306, 321, 473–474 Antigen, 20, 40, 42, 48, 62, 93, 103, 145, 172–176, 189, 229, 294, 298–299, 303, 318–319, 358–359, 387–388, 421–422, 503 Antigen-antibody interactions, 56, 173 Aptamers, 20, 55–65, 298 Autopsy, 427, 432

B Bacterial infections, 130, 186, 241, 321–323, 509 Bacteriocin typing, 231 Biochips, 10, 222, 383–391, 505–506 Biodefence, 483 Biological weapons, 484–490, 495–500

Biomarkers, 13–14, 17–23, 26, 102, 166, 204–206, 251, 287–307, 357, 359, 362, 395, 408, 510–511 Biomedical research, 2, 8, 34, 85, 104–106, 206, 263, 347, 406, 417, 498 Biopharmaceuticals, 40–41, 48, 414–417, 419–423 Biopharming, 41–42 Bioreceptors, 212 Biosensor, 61, 209–223, 280–283, 387, 390, 469, 473, 477–478, 506 Bioterrorism, 5, 203, 217, 483–484, 497 Biotyping, 228, 230–231, 310 Bioweapons, 484, 490, 496–497 BRCA1, 4, 115, 144, 290, 294, 300, 343–344, 403, 406, 445 C Cancer, 5, 90, 92, 102, 293, 297–302, 304, 306, 343, 356–360, 389, 419–420, 448–449, 509–510 Cancer biomarkers, 20, 293, 300, 389 Candidate markers, 104, 257, 295, 355 Cardiovascular markers, 505 CAT, 73, 75, 78, 80 CDNA cloning, 35 CDNA libraries, 42–43, 186, 190–192 CDNA sequences, 35, 141 CFTR, 45, 336–337 Chromosome, 1, 13, 38, 42, 50, 72, 87, 91–92, 116–117, 158–166, 332, 334–335, 339, 349, 353–354, 361, 367–369, 373–374, 389, 406, 429–430, 462 Chromosome painting, 159, 165 Clinical diagnostics, 2–3, 18, 81, 94, 147, 206, 512 Clinical forensic medicine, 432–433 Clinical medicine, 94–95, 113–114, 173, 222, 386–387, 426

M. Debnath et al., Molecular Diagnostics: Promises and Possibilities, C Springer Science+Business Media B.V. 2010 DOI 10.1007/978-90-481-3261-4,

515

516 Clinical testing, 4, 282, 335, 446 Clone, 35–36, 40, 43–44, 46–47, 58–59, 131, 139, 147, 183–184, 188–191, 201, 263, 295, 321, 325, 389 Cloning allergens, 190–191 Colorectal cancer, 90, 205, 303, 343, 348, 387, 440, 443 Combinatorial library, 57–58 Comparative genome hybridization, 197, 337, 343 Complex diseases, 23, 101, 256, 347–375, 400, 402 Computational tools, 16, 24, 92, 298 Cystic fibrosis, 4, 45, 88, 93, 115–116, 121, 144–145, 216, 222, 336–338, 341, 343, 350, 423, 514 Cystic Fibrosis Transmembrane Conductance Regulator gene, 331 Cytochrome P450, 273, 390, 405, 439–442 Cytogenetics, 153, 157, 159, 162–164, 334, 337, 348 D Dendrimers, 269, 271 Detections, 477 Diabetes, 9, 23, 26, 34, 88–89, 100, 103, 341–342, 348, 363–364, 367, 406, 418 Diagnosis, 3–5, 18, 20, 28, 49–51, 86–87, 89, 97–106, 111–112, 144–146, 164–166, 177–178, 203–204, 213, 216–217, 228–229, 232, 257, 276, 291, 294, 310–323, 331–344, 348–350, 352–360, 386–388, 417, 470, 509–510 Diagnostics Diagnostic medicine, 3 Diagnostic test, 2, 4–6, 9, 124, 174, 210, 229, 238–241, 302, 310–311, 319–321, 334, 337, 341–343, 366, 395, 449–450, 506, 509–512, 514 DNA aptamers, 56, 59–60, 62, 64–65 biosensor, 209–223 diagnosis, 331, 348 diagnostics, 50, 209, 213, 417 fingerprint, 45, 201, 233, 428–430, 457–458 fingerprinting, 201, 233, 428–430, 458 methylation, 431, 506 profiling, 324, 455–456, 461 selection, 55–65 sequencing, 36, 46, 87, 92, 130, 145, 343, 437 typing, 136, 455–459, 460, 463–464

Index Drug delivery, 262–263, 265, 267–270, 274–275, 280–283, 408 design, 91, 184, 191, 394, 406 discovery, 10–11, 20, 76–77, 98–99, 186–187, 190, 197, 202–204, 256, 288, 390, 396–399, 407–409, 438–439, 444–448, 506 response, 16, 23, 29, 106, 200, 202–203, 374, 385, 398–399, 402, 404–405, 407, 409, 436–438, 441, 449, 451–452, 513 screening, 81, 217, 282 Duchenne muscular dystrophy, 90, 336, 338 E Electrochemical biosensors, 214, 216–218 ELISA, 73, 77–78, 173–178, 213, 306, 321, 323, 473–474, 487–488 End labeling, 156 Environment monitoring, 209 Environmentome, 103 Enzyme-linked immunosorbant assay, 173 Epitope Mapping, 185–186, 188 Ethical legal social issues, 393 Ethics, 111–127 Expressed sequence tags (ESTs), 124 Expression of genes, 44, 143, 161 profile, 12, 14–17, 23, 28, 199–200, 204–206, 363, 390, 506 signature, 11 F Factor V (Leiden) mutation, 342 Fingerprint, 4, 19, 45, 118, 200–201, 232–233, 291, 324–325, 347, 428–430, 456–458, 463, 472 FISH, 10, 14, 74, 121, 156–160, 162–166, 249, 258, 299, 337, 470, 506 Fluorescence, 8, 10, 18, 64, 75, 81, 141, 145, 156–159, 161–162, 164–165, 174, 196, 210–211, 229, 249, 278, 299, 391, 476, 487 Food allergens, 469–470 borne disease, 468, 470–471, 479 diagnostics, 467–481 safety, 219, 280, 467–469, 471–473, 479–481 Forensic, 2, 4, 10, 45, 143, 147, 217, 278, 365, 425–433, 455–464, 506 Forensic medicine, 4, 425–433, 462

Index Functional genomics, 5, 13, 86, 98, 101–102, 186, 194, 201, 393 Fungal infections, 323 G GC-content, 133 Gene cloning, 36, 413–415 expression, 8, 12–17, 24–25, 49, 62–63, 73–76, 79, 80–82, 103, 106, 139, 161, 166, 194–197, 200–206, 245–246, 262, 272, 275, 336, 371, 384, 386, 390, 403 sequence, 14, 43, 86, 95, 123, 183, 279, 401, 414, 416, 474, 484 technology, 49, 82, 391, 455–464 therapy, 45, 50, 72, 81–82, 90, 106, 119–120, 125, 245, 270–272, 367, 398, 405, 414–415, 422, 510 transcript, 14, 82 transfer, 47, 51, 106, 125 variation, 395, 437, 443 Genetically modified organism, 124, 468 Genetic assay, 3, 113, 257 Genetic disease, 9, 49–50, 85, 99, 116, 119, 126, 144, 201, 270, 282, 331, 333–334, 336–343, 348–349, 438 Genetic disorders, 49, 114, 120, 127, 130, 162, 331–344, 394, 421, 506 Genetic markers, 9, 93, 202, 288, 359, 364, 374, 400–401, 442, 456–459 Genetic test, 3, 6, 50, 94–95, 111–115, 120–124, 126, 209, 334–336, 343–344, 350, 357, 364–365, 374–375, 390, 400, 405, 407–409, 436, 442, 450, 513 Genetic testing, 3, 6, 95, 113–115, 121–123, 332, 334–336, 343–344, 357, 374–375, 407–409, 439, 450, 513 Genetic testing diagnostics, 3, 6, 113–114, 144, 334, 343, 374, 450 Genome cloning, 35–36 DNA libraries, 42 medicine, 95 patents, 123–124 technology, 95, 123–126 Genomics, 5, 12–15, 21, 23, 25–26, 86, 94, 98, 101–102, 104, 186, 194, 245–247, 254, 282, 288, 375, 390, 404, 406–407, 415–416, 436, 449, 499–500, 507 Genosensors, 214–215 Genotyping, 15, 24, 136, 145–146, 237, 266, 314, 323, 325–326, 343–344, 366–367, 398–399, 407–408, 445, 450–451, 472

517 Germ line therapy, 119 GFP, 73–74, 76–77, 81 Gleevec, 15, 300, 302, 398, 406 Glycosylations, 39–40, 49, 250 Glycotherapeutics, 421 GUS, 73, 79, 129, 214, 489–490 H Haemophilia, 90, 336, 338, 365–367 Haemophilia A and B, 336 Hamster cell lines, 40, 48 Haplotype, 95, 288, 290, 339–340, 355, 371, 446 HapMap, 95 Health care, 4–6, 47, 85–86, 106, 112, 114, 120, 122, 254–257, 375, 404–405, 413–423, 439, 444, 449, 506 Hemochomatosis, 342 Herceptin, 300, 343, 398, 401, 405–406, 450 HGH, 48, 77, 238 HGP, 85–96, 112–114, 117–121, 126, 209 High throughput technology, 11 HUGO, 86 Human cell lines, 40–41, 49 Human genome project, 14, 17, 26, 85–87, 89, 92–95, 98–99, 112, 123–126, 143, 249–251, 347, 400, 428, 446, 500 Human Monoclonal Antibodies, 421 Humulin, 34 Hybridization, 7, 56, 142, 145–146, 153–166, 197, 201, 204, 213–218, 220, 237, 240, 276, 299, 301, 311–312, 319, 324–325 Hybridoma cell lines, 40 I Immobilisation, 214 Immunoassays, 3, 171–178, 212–213, 417, 469, 474, 487, 495, 506, 510–512 Immunogenicity, 39, 60, 64, 175, 177, 270 Immunological method, 173 Immunomagnetic separation, 473–475 ImmunoRCA, 174 Immunosensor, 219 Immunostimulatory vaccines, 496 Individualised drug treatment, 91 Inducer, 74–75 Infections, 40–41, 51, 62, 130, 144, 177, 186, 233, 237, 274, 310–323, 394, 439, 470–471, 487, 506 Infectious disease, 4–5, 7–9, 41, 48, 50–51, 100, 104, 144–145, 203, 217, 228, 240, 309–311, 324–328, 484–485, 508–509, 513–514

518 Inherited disorder, 44–45, 85, 332, 508 Insect cell lines, 40 In situ hybridization, 10, 142, 145, 153–166, 299, 301, 337, 506 K Kary Mullis, 129 L Lac, 78, 200 LacZ, 74, 78–79 Luc, 73, 75–76 M Marfan syndrome, 350 Market drivers, 508 Market Forecasts, 505 Mass spectrometry, 17–20, 22, 25, 101, 173, 216, 246, 249–252, 256–258, 301, 306, 464 Melting temperature, 133–134, 215 Metabolic profiling, 22 Metabolome, 12, 21–23, 25, 98 Metabolomics, 12, 21–23, 25–28, 99, 101–103, 194, 406 Microarray, 5, 8–9, 13, 16–17, 20, 24–25, 98, 166, 175, 193–206, 238, 282, 298, 301, 343–344, 384–386, 388–389, 391, 407, 437 Microbiological testing, 227–241, 479 Microsatellite, 146, 349, 365–366, 430, 446, 461 Mimetics and peptidomimetics, 421 Miniaturization, 10, 81, 95, 210, 219, 222, 282, 386–388 Mitochondrial DNA, 100, 430, 461–462 Mitochondrial DNA analysis, 430, 461 Molecular diagnostic industry, 6, 401–402 Molecular diagnostics, 4–10, 15, 75, 99, 114, 144–145, 204–205, 217–218, 228–230, 237, 239–241, 276, 310–311, 313, 317, 337, 342–343, 386, 390, 394, 407, 431, 451–452, 468, 506–507, 509, 512, 514 Molecular diagnostics segments, 505 Molecular epidemiology, 324–325 Molecular farming, 419–420, 422–423 Molecular imaging, 81, 275, 308 Molecular medicine, 9, 51–52, 119, 272–273, 360 Molecular methods, 164, 234–236, 314–316, 323–327, 372, 497

Index Molecular signature, 4, 6, 15, 204, 289, 297, 403, 509 Molecular techniques, 232, 236–237, 316, 319, 323, 326 Molecular tests, 4, 9, 228, 239, 241, 309–310, 323, 325–326, 514 Monogenic disorders, 332, 348, 400 MonoLEX, 60 MtDNA, 430, 459, 461–462 Multifactorial disorders, 332, 348–349 Multigenetic diseases, 104 Multiple endocrine neoplasia, 332, 341–342, 357 Multiple sclerosis, 348, 360–363 Mutations, 3, 24, 44–45, 49–50, 90, 93, 102, 104–105, 115, 136–137, 144–145, 215–216, 237–238, 290, 310, 314–315, 326, 332–333, 336–344, 347–351, 387–388, 400, 403, 406 Mycotoxins, 469, 474, 488–489, 491, 494, 496 N Nanocarrier, 192, 264, 267–271 Nanochips, 337 Nanodiagnostics, 276–278 NanoDLSA, 175 Nanofabrication, 222 Nanoparticles, 214, 216, 262–268, 270, 274–275, 391, 408 Nanosensor, 216, 477 Nanospheres, 268, 271 Nanotechnology, 10, 216, 256, 261–276, 278, 280–283, 386–387, 406, 506 Nanotube, 214, 262, 265, 270, 273–274, 279, 281–282 Non PCR based method, 459 Nucleic acid hybridization, 50, 214, 217–218, 319 Nucleic acid probe, 43, 140, 142, 154, 212, 233, 311, 325, 509 Nutrigenomics, 26, 370 O Obesity, 26, 95, 100–101, 104, 347–348, 367–370 Oligomers, 56 Oligonucleotide, 50, 56–60, 134, 137, 140, 154–156, 174, 195–197, 202, 204, 214–215, 217, 262, 271–272, 310, 324, 337, 384, 388 Oligonucleotide library, 57

Index Omics, 11–29, 101–102, 287, 406 Oncology, 4, 6, 16, 163–164, 173, 266, 343, 445, 510 P Patent, 9, 49, 105, 117, 123–126, 239, 408–409, 417–418, 423, 511 Pathogens, 42, 61–62, 146, 165, 210, 217–219, 230, 232–234, 236–238, 257, 311–314, 468–478, 484, 497 Pathology, 1–3, 19, 21, 25–26, 50, 166, 199, 204, 288, 295, 307, 362, 398, 400, 426 PCR, 7–8, 44, 131–147, 238–241, 294, 313, 326, 337–340, 366, 429–430, 456–457, 459–463, 474–476 PCR based methods, 324, 459–463 Peptide nucleic acid, 161, 165, 215 Personalised medicine, 206, 393–410 Phage display, 17–18, 181–191, 274, 421 Pharmaceutical industry, 10, 52, 63, 104–105, 171, 266, 268, 278, 356, 394–396, 398, 413–416, 418, 423, 448–449 Pharmaceuticals, 27, 33–52, 64, 87, 122, 398, 414–418 Pharmacogenetics, 6, 91, 387, 394, 405, 441, 444–445, 447–450, 506 Pharmacogenomics, 6, 86, 102, 106, 202–203, 326, 390, 394–395, 399, 403–407, 435–453, 508 Pharmacogenomic tests, 401, 439, 442–444, 509 Phenylketonuria, 336, 339, 374 PNA, 10, 161, 165, 215 Point of Care Diagnostic Testing, 505 Polymerase chain reaction, 1, 7, 38, 44–45, 129–148, 228, 233–234, 310–311, 317, 319–320, 359, 366, 387–388, 429–430 Polymerisation, 138 Polymorphism, 116, 137, 141, 218, 233, 301, 338, 355, 359, 364–366, 386–387, 430, 452, 457, 461–462 Post genomic, 97–106, 246, 307, 375, 384 Post mortem, 373, 433 Post translational modifications, 41, 289, 362, 375 Predisposition, 2–3, 5, 9, 89, 115, 117, 201, 276, 325, 332–333, 349–351, 386, 395, 400, 506, 513 Primer Design, 133 Probe, 5, 7–8, 43–44, 147, 153–161, 201, 214–217, 233, 237, 279, 311–312, 315, 324–325, 339, 384, 391, 418, 430, 457

519 Profiling microarray, 8, 20, 25, 166, 200, 359, 386 Prognostic, 4, 6, 10, 16, 23, 28, 50, 100, 166, 204–206, 256, 343, 355, 399 Promoter, 16, 43, 45, 47, 51, 58, 71–75, 80–81, 194, 321, 469 Promoter assay, 80 Protein arrays, 20–21, 205, 253, 306, 385 Protein microarray, 17, 20, 197–199, 205, 256, 495, 511 Protein microchips, 177 Protein profiling, 18, 20, 25, 251, 375 Protein – protein interaction, 17, 19, 21, 25, 77, 81, 217, 249, 257–258, 289 Proteome, 5, 12, 17–21, 25, 27, 98, 102, 184, 205, 246, 250–253, 257–258, 375, 389 Proteomics, 5, 12, 17–21, 26–28, 99, 101–105, 246–259, 291, 297–298, 307 Q QTL, 103, 351, 353, 367–369 Quantitative trait locus, 351 Quantum dots, 262, 265–266, 275, 280, 294, 408 R Radioimmunoassay, 172 Real-Time PCR, 59, 140–141, 146–147, 236–238, 337 Recombinant DNA, 33–52, 124–125, 182, 413–415, 469, 499 DNA pharmaceuticals, 33–52 DNA technology, 34–38, 47–51, 413–414, 499 protein, 34, 39, 41–42, 46, 48–49, 245, 393, 414–415, 422 Reporter gene assay, 72, 74–75, 80–81 Reporter, 7, 71–82, 140–141, 175, 217, 221 Reporter gene construct, 73, 81 Resistogram, 231 Restriction fragment length polymorphism testing, 430 Retinoblastoma, 45, 163, 290, 331, 357 RFLP, 145, 147, 232, 315, 337–340, 365–366, 459–460, 462 Ribotyping, 232, 324, 497 RNA, 1, 3, 6–7, 12–13, 16–17, 36–38, 46, 58–60, 105, 135–136, 145, 154, 162, 196, 238, 290–291, 320, 359, 384, 415, 431, 468, 475, 477, 506 RNA aptamers, 59–65

520 RT-PCR, 16, 44, 130, 137, 139, 141–142, 145, 233, 235, 291, 319 Ruc, 75–77

S Screening, 3, 6, 21, 43–44, 81, 102, 104, 115, 121, 130, 145, 184, 190–191, 198, 205, 218, 222, 230, 251, 255, 294, 300, 302, 314, 317, 334, 349, 358–359, 364, 366, 391, 394, 396, 399–400, 403, 406, 415, 417, 423 SELEX, 57–62, 64 Sensor technology, 215, 220–221, 476–478 Sequencing functional genomics, 5, 13, 86, 98, 101–102, 186, 194, 201, 393 Sex determination, 146, 161, 163, 429–430 Sexually transmitted diseases, 317, 418, 509 Signaling, 14, 17, 216, 249, 267, 348, 359, 372, 386 Simplex diseases, 350–351 Single gene disease, 88–90, 374 Single gene disorders, 89–90, 332 Single locus probe, 430 Single nucleotide polymorphism, 10, 24, 89, 116, 123, 137, 140, 201, 218, 266, 290, 301, 332, 365–366, 386, 389, 394–395, 436, 443, 445, 451–452, 462 Site specific mutation, 130 SLP, 315, 430 SNP, 10, 24, 89, 116, 123, 201, 266, 290, 301, 332, 337, 343, 349, 355, 366, 386, 389, 399–400, 407, 437, 443, 445, 452, 462 Somatic gene therapy, 119–120 STR, 430, 457, 461–462, 464 Systems biology, 11–13, 19, 25–27, 92, 217, 258, 307

Index T Taq polymerase, 129, 132 Template, 16, 38, 42, 58, 130, 132–135, 137–138, 140–141, 147, 174, 273–274 Thalassaemia, 336, 339 Therapeutics, 2, 5–6, 10, 28, 61, 63–64, 99, 175, 184, 187, 262–263, 267–289, 288, 394, 398, 402, 405–408, 414, 441–442, 486, 506, 509 Thermus auquaticus, 129 Tm , 129, 133–134, 514 Toxicity, 1–2, 10, 22–23, 86, 106, 200, 202, 204, 212, 220, 256, 264, 268–269, 275, 326, 398–399, 405, 407, 440, 443, 449 Toxicogenomics, 25 Transcriptional factors, 193 Transcriptomics, 12, 15–17, 23–27, 99, 103, 105, 194 Tumor markers, 102, 257, 357–358, 386, 406, 509 V Vaccines, 47–49, 51, 99, 184, 187–188, 398, 414–415, 417–422, 439, 485–486, 491, 496, 497, 499 Validation, 3, 98, 171, 205–206, 239–241, 291, 296–298, 301, 307, 315, 327, 441, 444, 463, 471, 513 Vector borne diseases, 309 Vectors, 36, 38, 42–43, 45, 47, 76, 79, 81–82, 106, 138, 143, 182, 186–187, 269–271, 389, 414, 422, 485 Viral infections, 313, 318–321 Virions, 183 X X-gal, 74, 78–79 X-linked inheritance, 348 Y Yeast two hybrid analysis, 17, 74

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